DNA replication of prokaryotes

FIS proteins

The Fis protein is a nucleoid associated protein that has previously been reported to act negatively in initiation of replication in Escherichia coli. In this work we have examined the influence of this protein on the initiation of replication under different growth conditions using flow cytometry. The Fis protein was found to be increasingly important with increasing growth rate. During multi-fork replication severe under-initiation occurred in cells lacking the Fis protein; the cells initiated at an elevated mass, had fewer origins per cell and the origins were not initiated in synchrony. These results suggest a positive role for the Fis protein in the initiation of replication. 

Fis (Factor for Inversion Stimulation) regulates many genetic systems. 2 

Another negative regulator of initiation in E. coli, the Fis protein, associates with oriC throughout most of the cell cycle; 3 similar to SeqA, Fis negatively influences replication initiation by regulating the occupation of DnaA on low-affinity sites (Cassler et al., 1995; Ryan et al., 2004). Fis specifically binds to a single site that is located between R2 and R3, and overlaps with the C3 DnaA binding site (Figure 1) 






Figure 1. A model of initiation replication and its regulation in E. coli by origin binding proteins (oriBPs). Large panel presents assumed sequence of events during the replication initiation and roles of particular oriBPs. The unwound DUE is accessible to the replication proteins complex (e.g., helicase DnaB, primase, and DNA Pol III). Small panel shows additional oriBPs divided in two subgroups, those involved in alternative scenarios that may occur under environmental stress conditions (upper part of the panel) and others, including those of unknown function (bottom part of the panel). Triangles' directions represent orientations of DnaA binding sites. Nucleotide bound status of DnaA is represented by blue and violet incomplete circles. Small arrows below gene names indicate gene orientations. In the small panel, different types of vertical lines represent type of action, activation (arrow), inhibition (bar-headed line) or unknown (question mark line). Horizontal lines indicate unspecific binding to oriC.


(Gille et al., 1991; Filutowicz et al., 1992). Fis binding is thought to competitively inhibit the interaction of DnaA with this region (Ryan et al., 2004), and Fis exhibits a DNA-bending activity that plays a yet-unknown role (Finkel and Johnson, 1992; Ryan et al., 2004).
In addition to competing with DnaA for binding to oriC, both Fis and SeqA also negatively regulate the interaction of another oriBP, IHF, with the origin. In contrast to the former two proteins, IHF positively regulates replication initiation (Hwang and Kornberg, 1992;Grimwade et al., 2000; Ryan et al., 2002). As the time of initiation draws near, increasing levels of DnaA trigger the displacement of Fis and the full methylation of DNA weakens SeqA binding, ending the repressive activities of these proteins (Slater et al., 1995; Ryan et al., 2004). The release of SeqA reveals the IHF binding site; displacement of Fis promotes IHF binding; and IHF binding leads to bending of the DNA (Polaczek, 1990; Cassler et al., 1995; Rice et al., 1996; Weisberg et al., 1996; Swinger and Rice, 2004). IHF then stimulates the binding of DnaA-ATP to low-affinity sites (thus redistributing the DnaA protein) and induces the unwinding of oriC (Grimwade et al., 2000). Notably, the transcription of the dnaA gene is also subject to regulation by the SeqA protein (Campbell and Kleckner, 1990;Theisen et al., 1993; Bogan and Helmstetter, 1997). Thus, the increased DnaA concentrations that trigger the displacement of Fis displacement presumably reflect the earlier release of the dnaA promoter from inhibition by SeqA.

1) http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083562
2) https://schneider.ncifcrf.gov/paper/fisinfo/fisinfo.pdf
3) http://journal.frontiersin.org/article/10.3389/fmicb.2014.00735/full

IHF binding proteins

The fold of IHF  is essentially the same as that of HU . The subunits of IHF, IHFα and IHFβ, are intertwined to form a body with two long β sheet arms  that extend from it . The arms interact exclusively with the minor groove of DNA and wrap around it. Each IHF subunit contains 5 Beta-sheets(S)  and 3 Alpha-Helices(H)  .The order of S and H is H1-H2-S1-S2-S'2-S'3-S3-H3 . The majority of the bending occurs at two kinks 9 base pairs(bp) apart and proline residues at the tip of the arm intercalates between base pairs  .

The phosphate backbone contacts 26 positively charged side chains  and interacts with the N-termini of all six helices  of the heterodimer. The ends of H1 and H3 form a clamp  by binding to opposite sides of the minor groove with respect to the intercalating proline. H2 forms a hydrogen bond from the bottom of the protein to adjacent DNA fragments.

Several Proteins Are Required for DNA Replication at the Replication Fork


Figure 11.7 provides an overview of the molecular events that occur as one of the two replication forks moves around the bacterial chromosome, and Table 11.1 summarizes the functions of the major proteins involved in E. coli DNA replication. 





Let’s begin with strand separation. To act as a template for DNA replication, the strands of a double helix must separate. The function of DNA helicase is to break the hydrogen bonds between base pairs and thereby unwind the strands; this action generates positive supercoiling ahead of each replication fork. As shown in Figure 11.7, an enzyme known as a topoisomerase (type II), also called DNA gyrase, travels in front of DNA helicase and alleviates positive supercoiling. After the two parental DNA strands have been separated and the supercoiling relaxed, they must be kept that way until the complementary daughter strands have been made. What prevents the DNA strands from coming back together? DNA replication requires single-strand binding proteins that bind to the strands of parental DNA and prevent them from re-forming a double helix. In this way, the bases within the parental strands are kept in an exposed condition that enables them to hydrogen bond with individual nucleotides.The next event in DNA replication involves the synthesis of short strands of RNA (rather than DNA) called RNA primers. These strands of RNA are synthesized by the linkage of ribonucleotides via an enzyme known as primase. This enzyme synthesizes short strands of RNA, typically 10 to 12 nucleotides in length. These short RNA strands start, or prime, the process of DNA replication. In the leading strand, a single primer is made at the origin of replication. In the lagging strand, multiple primers are made. The RNA primers are eventually removed. A type of enzyme known as DNA polymerase is responsible for synthesizing the DNA of the leading and lagging strands. This enzyme catalyzes the formation of covalent bonds between adjacent nucleotides and thereby makes the new daughter strands. In E. coli, five distinct proteins function as DNA polymerases and are designated polymerase I, II, III, IV, and V. DNA polymerases I and III are involved in normal DNA replication, whereas DNA polymerases II, IV, and V play a role in DNA repair and the replication of damaged DNA. DNA polymerase III is responsible for most of the DNA replication. It is a large enzyme consisting of 10 different subunits that play various roles in the DNA replication process ( Table 11.2 ).



The α subunit actually catalyzes the bond formation between adjacent nucleotides, and the remaining nine subunits fulfill other functions. The complex of all 10 subunits together is called DNA polymerase III holoenzyme. By comparison, DNA polymerase I is composed of a single subunit. Its role during DNA replication is to remove the RNA primers and fill in the vacant regions with DNA. Though the various DNA polymerases in E. coli and other bacterial species vary in their subunit composition, several common structural features have emerged. The catalytic subunit of all DNA polymerases has a structure that resembles a human hand. As shown in Figure 11.8 the template DNA is threaded through the palm of the hand; the thumb and fingers are wrapped around the DNA. 


FIGURE 11.8 The action of DNA polymerase. (a) DNA polymerase slides along the template strand as it synthesizes a new strand by connecting deoxyribonucleoside triphosphates (dNTPs) in a 5ʹ to 3ʹ direction. The catalytic subunit of DNA polymerase resembles a hand that is wrapped around the template strand. In this regard, the movement of DNA polymerase along the template strand is similar to a hand that is sliding along a rope. (b) The molecular structure of DNA polymerase I from the bacterium Thermus aquaticus. This model shows a portion of DNA polymerase I that is bound to DNA. This molecular structure depicts a front view of DNA polymerase; part (a) is a schematic side view.


The incoming deoxyribonuleoside triphosphates (dNTPs) enter the catalytic site, bind to the template strand according to the AT/GC rule, and then are covalently attached to the 3ʹ end of the growing strand. DNA polymerase also contains a 3ʹ exonuclease site that removes mismatched bases, as described later. As researchers began to unravel the function of DNA polymerase, two features seemed unusual 



FIGURE 11.9 Unusual features of DNA polymerase function.
(a) DNA polymerase can elongate a strand only from an RNA primer or existing DNA strand. (b) DNA polymerase can attach nucleotides only in a 5ʹ to 3ʹ direction. Note the template strand is in the opposite, 3ʹ to 5ʹ, direction.

DNA polymerase cannot begin DNA synthesis by linking together the first two individual nucleotides. Rather, this type of enzyme can elongate only a preexisting strand starting with an RNA primer or existing DNA strand (Figure 11.9a). A second unusual feature is the directionality of strand synthesis. DNA polymerase can attach nucleotides only in the 5ʹ to 3ʹ direction, not in the 3ʹ to 5ʹ direction (Figure 11.9b). Due to these two unusual features, the synthesis of the leading and lagging strands shows distinctive differences (Figure 11.10 ).


FIGURE 11.10 The synthesis of DNA at the replication fork.

The synthesis of RNA primers by primase allows DNA polymerase III to begin the synthesis of complementary daughter strands of DNA. DNA polymerase III catalyzes the attachment of nucleotides to the 3ʹ end of each primer, in a 5ʹ to 3ʹ direction. In the leading strand, one RNA primer is made at the origin, and then DNA polymerase III can attach nucleotides in a 5ʹ to 3ʹ direction as it slides toward the opening of the replication fork. The synthesis of the leading strand is therefore continuous. In the lagging strand, the synthesis of DNA also elongates in a 5ʹ to 3ʹ manner, but it does so in the direction away from the replication fork. In the lagging strand, RNA primers must repeatedly initiate the synthesis of short segments of DNA; thus, the synthesis has to be discontinuous. The length of these fragments in bacteria is typically 1000 to 2000 nucleotides. In eukaryotes, the fragments are shorter—100 to 200 nucleotides. Each fragment contains a short RNA primer at the 5ʹ end, which is made by primase. The remainder of the fragment is a strand of DNA made by DNA polymerase III. The DNA fragments made in this manner are known as Okazaki fragments, after Reiji and Tuneko Okazaki, who initially discovered them in the late 1960s. To complete the synthesis of Okazaki fragments within the lagging strand, three additional events must occur: removal of the RNA primers, synthesis of DNA in the area where the primers have been removed, and the covalent attachment of adjacent fragments of DNA (see Figure 11.10 and refer back to Figure 11.7). In E. coli, the RNA primers are removed by the action of DNA polymerase I. This enzyme has a 5ʹ to 3ʹ exonuclease activity, which means that DNA polymerase I digests away the RNA primers in a 5ʹ to 3ʹ direction, leaving a vacant area. DNA polymerase I then synthesizes DNA to fill in this region. It uses the 3ʹ end of an adjacent Okazaki fragment as a primer. For example, in Figure 11.10, DNA polymerase I would remove the RNA primer from the first Okazaki fragment and then synthesize DNA in the vacant region by attaching nucleotides to the 3ʹ end of the second Okazaki fragment. After the gap has been completely filled in, a covalent bond is still missing between the last nucleotide added by DNA polymerase I and the adjacent DNA strand that had been previously made by DNA polymerase III. An enzyme known as DNA ligase catalyzes a covalent bond between adjacent fragments to complete the replication process in the lagging strand (refer back to Figure 11.7). In E. coli, DNA ligase requires NAD+ to carry out this reaction, whereas the DNA ligases found in archaea and eukaryotes require ATP. Figure 11.11 shows how new strands are constructed from a single origin of replication. To the left of the origin, the top strand is made continuously, whereas to the right of the origin it is made in Okazaki fragments. By comparison, the synthesis of the bottom strand is just the opposite. To the left of the origin it is made in Okazaki fragments and to the right of the origin the synthesis is continuous.



FIGURE 11.11 The synthesis of leading and lagging strands outward from a single origin of replication.

Termination of DNA replication

DNA replication can be divided into three distinct steps: initiation, elongation, and termination. The bidirectional replication of a circular chromosome of bacteria terminates at a position where the two replication forks meet. Bacteria have  a system that ensures termination will occur within a restricted terminus region. This is achieved by a combination of a DNA motif of 20 to 30 bp, called the ter sequence, and a cognate termination protein that recognizes ter sites and binds to them tightly.

Termination Eventually, the two replication forks of the circular E. coli chromosome meet at a terminus region containing multiple copies of a 20 bp sequence called Ter (Fig. 25–18). The Ter sequences are arranged on the chromosome to create a trap that a replication fork can enter but cannot leave. The Ter sequences function as binding sites for the protein Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only one direction. Only one Tus-Ter complex functions per replication cycle—the complex first encountered by either replication fork. Given that opposing replication forks generally halt when they collide, Ter sequences would not seem to be essential, but they may prevent overreplication by one fork in the event that the other is delayed or halted by an encounter with DNA damage or some other obstacle. So, when either replication fork encounters a functional Tus-Ter complex, it halts; the other fork halts when it meets the first (arrested) fork. The final few hundred base pairs of DNA between these large protein complexes are then replicated (by an as yet unknown mechanism), completing two topologically interlinked (catenated) circular chromosomes (Fig. 25–19).


FIGURE 25–19 Role of topoisomerases in replication termination. Replication of the DNA separating opposing replication forks leaves the completed chromosomes joined as catenanes, or topologically interlinked circles. The circles are not covalently linked, but because they are interwound and each is covalently closed, they cannot be separated—
except by the action of topoisomerases. In E. coli, a type II topoisomerase known as DNA topoisomerase IV plays the primary role in the separation of catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break.

Eventually, the two replication forks of the circular E. coli chromosome meet at a terminus region containing multiple copies of a 20 bp sequence called Ter (Fig. 25–18).



The Ter sequences are arranged on the chromosome to create a trap that a replication fork can enter but cannot leave. The Ter sequences function as binding sites for the protein Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only one direction. Only one Tus-Ter complex functions per replication cycle—the complex first encountered by either replication fork. Given that opposing replication forks generally halt when they collide, Ter sequences would not seem to be essential, but they may prevent overreplication by one fork in the event that the other is delayed or halted by an encounter with DNA damage or some other obstacle. So, when either replication fork encounters a functional Tus-Ter complex, it halts; the other fork halts when it meets the first (arrested) fork. The final few hundred base pairs of DNA between these large protein complexes are then replicated (by an as yet unknown mechanism), completing two topologically interlinked (catenated) circular chromosomes (Fig. 25–19). DNA circles linked in this way are known as catenanes. Separation of the catenated circles in E. coli requires topoisomerase IV (a type II topoisomerase). The separated chromosomes then segregate into daughter cells at cell division. The terminal phase of replication of other circular chromosomes, including many of the DNA viruses that infect eukaryotic cells, is similar.

DNA termination of replication in E. coli & B. subtilis

Introduction

In prokaryotes such as E. coli and B. subtilis, chromosomal DNA exists in a circular fashion whereby DNA replication takes place at a common origin (oriC) ^[1]. Two replication forks move bidirectionally from oriC to replicate DNA until they eventually meet, and the forks fuse with one another to form two circular daughter chromosomes ^[2]. The region where the two replication forks meet is defined as the “terminus region”, located roughly opposite of oriC ^[3]. Bacteria use a “replication fork trap” system for successful termination of replication and fork fusion. This requires two factors:

- DNA terminator (Ter) sites
- A specific terminator protein that can bind Ter

DNA terminator (Ter) sites 5

Ter is a short consensus DNA sequence (around 20 base pairs long) that enables binding of its cognate terminator protein in order to arrest or halt replication fork progression in a polar manner i.e. it blocks replication fork coming in one direction (the non-permissive side) but allows passage when replication fork approaches from the other direction (the permissive side) ^[5]. In both E. coli and B. subtilis, multiple Ter sites are organized into two subgroups that flank the terminus region. Since replication fork arrest is unidirectional, Ter sites are distributed so that one subgroup only arrests the clockwise-moving fork while the other subgroup only arrests the anti-clockwise moving fork ^[4]. A suggestive reason for the presence of multiple Ter sites is to act as a safety measure to ensure termination of replication and fork fusion occur within the terminus region even if one of the replication forks managed to precede the innermost Ter sites.

DNA terminator proteins

DNA terminator proteins are proteins that can recognize and bind Ter DNA to form a complex in order to achieve polar trapping of replication forks ^[4]. In E. coli, this protein is called Tus (terminus utilization substance) whilst in B. sutilis, it is called RTP (replication termination protein). Based on experimental data using mutated Ter sites and DNA terminator mutants suggests that both protein-DNA (terminator-Ter) and protein-protein (terminator-replisome) interactions are important for successful polar fork arrest ^[6].

Biological roles of replication fork traps

As a matter of fact, it is not entirely essential to have replication fork traps in E. coli and B. subtilis to terminate DNA replication. This is because no deleterious effects were observed with respect to tus and rtp gene deletion experiments ^[4]. Therefore one possible reason of having replication fork traps is to reduce collisions of DNA replication and transcription apparatus as most genes are oriented in the origin to termination direction. Another role which replication fork traps may perform is to prevent over-replication of the chromosome by ensuring the two opposite replisomes dislodge within the termination complex.





The circular chromosomes of E. coli (left) and B. subtilis (right) showing their respective origin of replication (Ori C), direction of the two replication forks (red arrows) and their subsequent fork traps (blue and green). Modified from ^[4].

Ter sites

There are ten functional Ter sites (TerA-J) in E. coli, each 23 base pairs in length and are arranged in two opposite subgroups of five. No sequence symmetry or direct repeats occur across all Ter sites. For this reason, along with the fact that its cognate DNA terminator protein partner – Tus is asymmetric in nature, enables Tus to bind as a monomer. The core sequence of Ter is between base positions 6-19 and in particular, the G-C base pair at position 6 is strictly conserved ^[1][7]. These consensus sequences are important in Tus-Tercomplex formation.

Crystal structure of Tus when bound to Ter

The crystal structure of the Tus-Ter complex was first unraveled by Kamada and co-workers ^[5]. Tus is a 36 kDa protein that binds Ter as a monomer. It consists of two asymmetrical domains: a larger N-terminal domain and a smaller C-terminal domain. Both domains are classified as α+β structures, made up of α-helices (αI-V) and β-sheets (βA-O), and are linked by 4 long loops (L1, L2, L3 and L4) that separate in between. Ter binding motif occurs in the interdomain β cleft of Tus which connects between the N- and C- terminal domains. It composes of two twisted antiparallel β strands (βF-βG and βH-βI) containing lots of basic residues, hence is positively charged. This large interdomain cleft is responsible for base specific recognition of Ter and intercalates tightly into the major groove of DNA. Furthermore, asymmetry in Tus binding gives rise to polar replication fork arrest. α-helices protruding at two sides are biased and mainly make contacts at the non-permissive side. This helps to protect the interdomain β cleft from direct contacts with replisomal proteins, which would be enough to displace Tus at the permissive side. Overall Tus embraces 13 base pairs of the DNA duplex through sugar-phosphate backbone contacts mediated byhydrogen bonds (H-bonds) or van der Waal interactions, as well as base contacts via H-bonds responsible for Ter recognition. Whilst the α-helical regions clamp the DNA at a girth-like manner, Tus is stabilized by high affinity binding of the interdomain.

Mechanism of polar fork arrest

Two models were proposed for halting replication fork movement at the non-permissive or blockage end ^[5]:

The “interaction” model
The “clamp” model


Tof1 and Csm3 block the molecular ‘sweepase’ Rrm3 from removing Fob1 from Ter. 6
A. Tof1–Csm3 interacts with Mcms and travels with the replication fork. In wild-type S. cerevisiae, Tof1 and Csm3 function cooperatively to prevent Rrm3 from dislodging Fob1 from Ter. When the replisome approaches the non-permissive face of Fob1, Fob1 mediates arrest of the replication fork.
B. In cells deleted for tof1 or csm3, Rrm3 acts as a molecular ‘sweepase’ to remove Fob1 from chromosomal DNA. Polar arrest of a replication fork does not occur in these mutant cells.
C. In cells deleted for tof1 and rrm3 or csm3 and rrm3, Fob1 remains bound to Ter, and Fob1 mediates polar arrest of the replication fork.

The "Interaction" model

This model encapsulates the idea that since helicase DnaB is the first replisomal protein to encounter the Tus-Ter complex as it unwinds DNA in the 5’-3’ direction on the lagging strand, protein-protein interactions between DnaB and Tus may be involved in replication fork arrest ^[7]. In fact, based on site-directed mutagenesis studies, this special contact occurs at the L1 loop of Tus ^[8]. Besides, specificity of binding is elicited as some helicases in E. coli such as Rep is not blocked by Tus at the non-permissive end ^[6].

The "Clamp" model

The model as the name suggests, refers to phosphate backbone contacts of Tus via α-helical regions of N- and C-terminal domains that completely surround Ter at the non-permissive face,and this tight binding forms a physical barrier for stalling helicase DnaB thus fork progression ^[5]. Also, the idea was put forward that N-terminal α-helices can tangle the unwound 3’-5’ DNA which can strip off DnaB.
Furthermore based on base substitution experiments of Ter sites, it is demonstrated that exposure of the flipped Cytosine at position 6 of Ter - C(6) due to DnaB unwinding activity leads to binding within a hydrophobic pocket between Ile79 and Phe140 of Tus, forming a locked complex ^[7] which has a even higher binding affinity for Ter than normal double-stranded Ter binding. This conformation further enhances the stability of polar fork arrest and may act as a back-up system in case e.g. a DnaB-Tus-Terarrest mechanism fails ^[6].

Although there is evidence to support either model, possibly a more feasible and logical mode of replication fork arrest in E. coliis to employ both models as a “fail-safe mechanism” ^[6]. As the replication machinery approaches the Tus-Ter complex on the non-permissive side, interactions between DnaB and Tus should be sufficient to halt replisome progression and this serves as the primary fork arrest mechanism. In case where this first blockage fails, further unwinding of DNA into C(6) by DnaB leads to lock complex formation, giving Tus a second attempt to stop the replication fork.
On the other hand, at the permissive or passage end of Tus, absence of a α-helical barrier ^[2] as well as strand separation by DnaB causes progressive loss of Tus-Ter contacts ^[7]. As a result Tus can easily dissociate and the replisome passes through the Ter site.

In B. subtilis: RTP-Ter complex

Such systems were discovered in Escherichia coli and Bacillus subtilis, through the identification of the accumulation of Y-shaped replication intermediates at specific sites in the terminus regions (1), and they have been extensively characterized genetically and biochemically (2-4).


In summary, the termination sequences and replication terminator protein structures are dissimilar in E. coli and B. subtilis, indicating independent origin of evolution.


That means the same mechanism would have evolved twice independently. The termination mechanism of replication had to be existing and functional in the first living organism. If from there on it would have evolved  and diverged, the existing structures in bacteria should be similar, and possible to be traced back to a common ancestor. Since that seems not to be the case, the only alternative would be that the system arised twice in two separate origin of life events.  

Replication termination in E. coli and B. subtilis 





 The E. coli termination protein is encoded by the tus (terminus utilization substance) gene and has a molecular weight of 36 kDa (309 amino acid residues). The Tus protein specifically binds to the ter sites containing the consensus sequence of about 20 bp, and the Tus-Ter complex arrests a replication fork approaching from one direction but not from the other. This arrest is thought to be due to the orientation-dependent inhibition of unwinding of the DNA duplex by the DnaB DNA helicase at the apex of the replication fork (Fig. 1). Seven ter sites have been identified in the terminus region of the E. coli chromosome, as shown schematically in Figure 2. 






The clockwise replication fork can pass through the group1 ter sites, but if it arrives at the group2 ter sites before it meets the counterclockwise replication fork, it will stall there. Similarly, the counterclockwise replication fork will stall at the group1 ter sites if it has not met the clockwise fork. Thus, the termination event is regulated to occur in the terminus region opposite the oriC replication origin.


Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex 4

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. 

DNA replication in Escherichia coli initiates at oriC, the unique origin of replication, and proceeds bidirectionally (119). This creates two replication forks that invade the duplex DNA on either side of the origin. The forks move around the circular chromosome at a rate of about 1,000 nucleotides per second and so meet about 40 min after initiation in a region opposite oriC. In this region are located a series of sites, called termination or Ter sites, that block replication forks moving in one direction but not the other (Fig. ​(Fig.1).1). This creates a “replication fork trap” that allows forks to enter but not to leave the terminus region (66,67).








Replisome of E. coli and mechanism of replication fork arrest by a Tus-Ter complex. 
(A) The replisome moving along the DNA template approaches Tus, and the DnaB helicase assists primase to lay down the last lagging-strand primer. 
(B) DnaB helicase action isblocked by Tus, and DnaB dissociates from the template. 
(C) DNA polymerase III (Pol III) holoenzyme completes leading-strand synthesis up to the Tus-Ter complex and 
(D) synthesizes the last Okazaki fragment on the lagging strand, which will eventually be ligated by DNA ligase to the penultimate fragment following removal of its RNA primer by DNA polymerase I (not shown). (E) The holoenzyme then dissociates, leaving a Y-forked structure that is single stranded on the lagging strand near the Tus-Ter complex.


Replication Is Terminated When the Replication Forks Meet at the Termination Sequences

On the opposite side of the E. coli chromosome from oriC is a pair of termination sequences called ter sequences. A protein known as the termination utilization substance (Tus) binds to the ter sequences and stops the movement of the replication forks. As shown in Figure 11.13 , one of the ter sequences designated T1 prevents the advancement of the fork moving left to right, but allows the movement of the other fork (see the inset to Figure 11.13).




FIGURE 11.13 The termination of DNA replication. Two sites in the bacterial chromosome, shown with rectangles, are ter sequences designated T1 and T2. The T1 site prevents the further advancement of the fork moving left to right, and T2 prevents the advancement of the fork moving right to left. As shown in the inset, the binding of Tus prevents the replication forks from proceeding past the ter sequences in a particular direction.


The fork moving right to left, but allows the advancement of the other fork. In any given cell, only one ter sequence is required to stop the advancement of one replication fork, and then the other fork ends its synthesis of DNA when it reaches the halted replication fork. In other words, DNA replication ends when oppositely advancing forks meet, usually at T1 or T2. Finally, DNA ligase covalently links the two daughter strands, creating two circular, double-stranded molecules. After DNA replication is completed, one last problem may exist. DNA replication often results in two intertwined DNA molecules known as catenanes (Figure 11.14 ).


FIGURE 11.14 Separation of catenanes. DNA replication can result in two intertwined chromosomes called catenanes. These catenanes can be separated by the action of topoisomerase.


Fortunately, catenanes are only transient structures in DNA replication. In E. coli, topoisomerase II introduces a temporary break into the DNA strands and then rejoins them after the strands have become unlocked. This allows the catenanes to be separated into individual circular molecules.

The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. 2

Replication Termination in Escherichia coli: Structure and Antihelicase Activity of the Tus-Ter Complex 1

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. 

The presence of the fork trap constructs has several important and advantageous consequences for the organism in question. 7 These include:

Due to the high conservation of sequences within a species, the presence of multiple trap regions introduces a level of redundancy, whereby if a single base mutation in the terelement was to inactivate the region, another ter element further towards the terminus-to-origin direction might be used [2].

Multiple ter sites allow for a level of speed regulation, such that the faster of 2 replication forks might be slowed down when progressing faster than the other. This might occur if one side of the replicating chromosome had to pause to allow DNA repair mechanisms to be completed [12].


However these advantages do not explain the developmental pressures leading to the development of these systems individually, nor do they explain why the removal of activity of these sites by knockout causes no functional phenotype.
The functional significance of the replication fork trap construct is that without it, replication would not be forced to terminate at 180˚ from the origin, and it may continue back in the terminus-to-origin direction. The development of a fork trap construct in circular chromosomes suggests that this would be undesirable for the organism. Reasons for this may include the fact that the majority of transcribed and translated genes are oriented for transcription in origin-to-terminus direction. If replication machinery was allowed to continue on in a terminus-to-origin orientation, there would be the potential for head-on-collision between transcription and replication machinery, which has been proven in the past to have deleterious affects [6].
More recent studies have showed a highly important and genome wide regulatory role for the ter sites and their cognate binding proteins. Study of E. coli shows that when mutations or knockouts are introduced to DNA polymerase A, the loss of function of the ter sites leads to increased levels of DNA overproduction. Furthermore, cells with Tus-terB deletions also exhibited increased rates of DNA overproduction. When Tus protein was provided to such cells, this overproduction was corrected, confirming that the absence of Tus (and not the loss of polA function) was responsible for the DNA overproduction [8]. Similar studies in B. subtilis show that when mutations are introduced to partitioning genes in combination with mutation to the rtp gene, an increase in anucleate cell production results. Partitioning genes are genes responsible for the accurate separation of replication products into daughter cells, and include the proteins spoIIIE and ripX. B. subtilis studies show that whilst the loss of rtp does not cause partitioning defects in wild-type background, when combined with partitioning defects an increase in anucleate cell production results [9]. These studies suggest a more global role for the the ter sites and their cognate binding proteins, and suggests their global responsibility for maintainance of the termination of replication as a safeguard against the affects of mutations in the highly important replication machinery.

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1197808/
2) http://www.tandfonline.com/doi/pdf/10.4161/15384101.2014.958912
3) http://what-when-how.com/molecular-biology/termination-of-dna-replication-molecular-biology/
4) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1197808/
5) http://proteopedia.org/wiki/index.php/User:Meng_Han_Liu/DNA_termination_of_replication_in_E._coli_%26_B._subtilis
6) http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06656.x/full
7) http://proteopedia.org/wiki/index.php/Lauren_Fowler/Replication_termination_in_E._coli_and_B._subtilis

http://www.nature.com/nature/journal/v525/n7569/abs/nature14866.html

DNA damage and repair


Fate of the replisome following arrest by UV-induced DNA damage in Escherichia coli.  7

ABSTRACT

Accurate replication in the presence of DNA damage is essential to genome stability and viability in all cells. In Escherichia coli, DNA replication forks blocked by UV-induced damage undergo a partial resection and RecF-catalyzed regression before synthesis resumes. These processing events generate distinct structural intermediates on the DNA. However, the fate and behavior of the stalled replisome remains a central uncharacterized question. Here, we use thermosensitive mutants to show that the replisome’s polymerases uncouple and transiently dissociate from the DNA in vivo. Inactivation of α, β, or τ subunits within the replisome is sufficient to signal and induce the RecF-mediated processing events observed following UV damage. By contrast, the helicase–primase complex (DnaB and DnaG) remains critically associated with the fork, leading to a loss of fork integrity, degradation, and aberrant intermediates when disrupted. The results reveal a dynamic replisome, capable of partial disassembly to allow access to the obstruction, while retaining subunits that maintain fork licensing and direct reassembly to the appropriate location after processing has occurred.

The replisome consists of several, multisubunit protein complexes and is responsible for duplicating the genome. In Escherichia coli, it is comprised of three DNA polymerase complexes tethered to the DNA template by dimeric processivity factors, a τ complex that couples leading and lagging strand synthesis, and a helicase–primase complex that separates the duplex DNA and primes lagging strand synthesis (1–3).

When the replisome encounters DNA damage that blocks its progression, the potential for mutagenesis, rearrangements, and lethality increases significantly. Replication in the presence of DNA damage can generate mutations if the wrong base is incorporated, rearrangements if it resumes from the wrong site, or lethality if the obstructing lesion cannot be overcome. Following the arrest of replication at UV-induced damage, the nascent lagging strand is partially resected by the combined action of the 

RecQ helicase  In prokaryotes RecQ is necessary for plasmid recombination and DNA repair from UV-light, free radicals, and alkylating agents. This protein can also reverse damage from replication errors. 8 The RecQ family of helicases are enzymes that unwind DNA so that replication, transcription, and DNA repair can occur. 9
RecQ helicases are highly conserved from bacteria to man. 10, 11. Germline mutations in three of the five known family members in humans give rise to debilitating disorders that are characterized by, amongst other things, a predisposition to the development of cancer. One of these disorders — Bloom's syndrome — is uniquely associated with a predisposition to cancers of all types. So how do RecQ helicases protect against cancer? They seem to maintain genomic stability by functioning at the interface between DNA replication and DNA repair. 

RecJ nuclease (4, 5). The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair. 12

RecF-O-R, along with RecA, limit this degradation and promote a transient regression of the DNA branch point, which is thought to be important for restoring the damaged region to a form that can be acted on by repair enzymes or translesion DNA polymerases (4–10). These processing events generate distinct structural intermediates on the DNA, a technique that allows one to identify the shape and structure of DNA molecules (5, 11).

Although the processing that occurs on the DNA is well characterized, little is known about the behavior or composition of the replisome itself during these events. If the replisome remains bound to the arresting lesion, it may sterically obstruct repair or bypass from occurring. Conversely, complete dissociation of the replisome would likely abolish the licensing for the replication fork and expose DNA ends that have the potential to recombine, generating deletions, duplications, or rearrangements on the chromosome. Recent studies in vitro have suggested that dynamic interactions between replisome components may play a role in allowing the machinery to overcome specific challenges such as collisions with the transcription apparatus or DNA-bound proteins (1, 12, 13).  UV-induced photoproducts is a biologically relevant lesion that is known to block the progression of the replisome when located in the leading strand template (6,14–16). The results demonstrate that the DNA polymerases can dissociate from the replisome in a modular manner without compromising the integrity of the replication fork. Dissociation of the DNA polymerase from the replisome is sufficient and can serve to initiate the processing of the replication fork DNA via the RecF pathway, similar to that seen when replication is arrested by UV-induced damage. By comparison, the helicase complex remains associated with the replication fork throughout the recovery process. If the helicase is disrupted, aberrant intermediates, degradation, and loss of fork integrity ensue. We propose that the retention of the helicase is needed to maintain licensing for the replication fork and direct reassembly to the appropriate location after processing has occurred.


A schematic of each of the components of the replisome tested in this study and their function is presented in Fig. 1A. Temperature-sensitive mutants exist in subunits from each of replisome’s complexes for which viability or functionality is supported at 30 °C, but not at 42 °C (Fig. 1B). Although replication proceeds normally at the permissive temperature, it rapidly decreases following inactivation of the thermosensitive protein at the restrictive temperature, similar to that seen after UV irradiation (Fig. 1C). The exception to this is in the proofreading subunit ε, encoded by dnaQts, which is mutagenic at the restrictive temperature, but is not essential for viability or replication (17).





Replication is disrupted by UV-induced damage or following inactivation of the DNA polymerase, τ complex, or helicase–primase complex. 
(A) A diagram of the replisome, indicating the subunits of each protein complex. 
(B) Thermosensitive mutants that inactivate the polymerase core, τ complex, or helicase complex are viable at 30 °C but fail to grow at the restrictive temperature of 42 °C following overnight incubation. 
(C) The rate of DNA synthesis is inhibited following UV-induced damage or inactivation of the replisome’s essential subunits. Wild-type or mutant cultures, grown at 30 °C were pulse-labeled with 1 µCi per 10 µg/mL [³H]thymidine for 2 min at the indicated times following mock treatment (open symbols), 50 J/m² UV irradiation (filled symbols), or a shift to 42 °C (filled symbols). The amount of radioactivity incorporated into the DNA, relative to pretreated cultures is plotted. Error bars represent SE of two experiments.




Model of replisome at UV-induced damage. Upon encountering an arresting lesion (PD, pyrimidine dimer) 
(i), DNA synthesis becomes uncoupled and the polymerases transiently dissociate. 
(ii) This serves as a signal to initiate the replication fork DNA processing by the RecF-pathway gene products (gray circles) allowing repair enzymes (NER) or translesion polymerases to access the lesion. 
(iii) The helicase–primase complex remains bound to the template DNA and serves to maintain the licensing and integrity of the replication fork, directing replisome reassembly to the correct location once the lesion has been processed.



Cellular Characterization of the Primosome and Rep Helicase in Processing and Restoration of Replication following Arrest by UV-Induced DNA Damage in Escherichia coli  4

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion.

PriA, PriB, and PriC were originally identified as proteins required for replication of single-strand ϕX174 phage DNA in vitro and in vivo (70, 71). In vitro, the proteins function as a complex that is required for processive priming to occur behind the replicative helicase, DnaB (1, 2). PriA initially binds a hairpin structure on the ϕX174 chromosome, followed by PriB, DnaT, and PriC. The resulting complex then recruits DnaC, which loads the DnaB helicase onto the chromosome. The DnaG primase is then able to associate with DnaB to synthesize RNA primers. While DnaG and DnaB are sufficient for primer synthesis on ϕX174 DNA (1), specific and processive priming of single-stranded DNA binding protein-coated phage DNA requires PriA (2). In vivo, conversion of ϕX174 from its plus-strand form to its minus-strand replication intermediate requires PriA and other Escherichia coli host proteins (40). E. coli strains lacking PriA have reduced viability, growth rates, and culture densities relative to wild-type cells (36). priA mutants are also constitutively induced for the SOS response, and cells lacking PriA produce filaments extensively (49). Taken together, these observations led early researchers to propose that the primosomal proteins promote efficient priming for Okazaki fragments during lagging-strand replication (35, 38).


Replication forks must deal with a variety of obstacles that may impede their progress, including DNA-bound proteins, secondary structures, strand breaks, and adducts or damage to the DNA bases themselves. With respect to DNA base damage, UV irradiation with 254-nm light has often served as a model to address the question of how replication recovers following encounters with this form of impediment. UV irradiation induces two primary photoproducts, cis, syn-cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone photoproducts (6-4 PPs) (59, 67, 68). Although these lesions block DNA polymerases and arrest replication (28, 58), growing E. coli cultures survive doses that produce more than 2,000 lesions per genome (30), indicating that cells contain efficient mechanisms to process these lesions when they are encountered during replication.

The recovery of replication following arrest by UV-induced DNA damage occurs through a sequence of well-characterized steps. Following arrest, the nascent lagging strand is partially degraded by the combined action of the RecJ nuclease and RecQ helicase. This processing is thought to restore the lesion-containing region to a double-stranded form that can be accessed and repaired by the nucleotide excision repair complex (17). 

RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli 5

All cells must faithfully replicate their genomes in order to reproduce. However, if not repaired, DNA damage that blocks replication can lead to a loss of genomic stability, mutations, or cell death. Despite the importance of the process by which replication recovers, the cellular mechanism(s) by which this occurs in DNA repair proficient cells remains largely uncharacterized. Irradiation of cells with near UV light induces lesions in the DNA which block replication. In E. coli, replication is transiently inhibited following a moderate dose of UV irradiation, but it eficiently recovers following the removal of the UV-induced lesions . The eficient recovery of replication in wild-type cells is accompanied by the partial degradation of the nascent DNA at the replication fork prior to the resumption of DNA synthesis . However, it is not known whether this degradation is required for, or contributes in any way to, the normal recovery process. The resumption of replication following UV-induced DNA damage is largely dependent upon the removal of the lesions by nucleotide excision repair . However, a large body of work with repair-deficient mutants has shown that UV irradiation can lead to recombination events when replication forks encounter DNA damage that cannot be repaired. In these mutants, the recovery of replication is severely inhibited, resulting in loss of semiconservative replication, high frequencies of chromosomal exchanges, and extensive cell death . In contrast, these recombination events are eficiently suppressed in normal, repair-proficient cells; survival is greatly enhanced and the recovery of replication is much more eficient, suggesting that the normal mechanism of recovery may be quite di€fferent from that observed in repair-deficient mutants. In addition to removal of the lesions, however, the recovery of replication also requires the function of RecA and the recF pathway proteins Historically, because most of these proteins were identified through recombination 


RecQ helicase and RecJ nuclease provide complementary functions to resect DNA for homologous recombination 6

Recombinational DNA repair by the RecF pathway of Escherichia coli requires the coordinated activities of 


RecA, 
RecFOR, 
RecQ, 
RecJ,  
single-strand DNA binding (SSB) proteins.


These proteins facilitate formation of homologously paired joint molecules between linear double-stranded (dsDNA) and supercoiled DNA. Repair starts with resection of the broken dsDNA by RecQ, a 3′→5′ helicase, RecJ, a 5′→3′ exonuclease, and SSB protein. The ends of a dsDNA break can be blunt-ended, or they may possess either 5′- or 3′-single-stranded DNA (ssDNA) overhangs of undefined length. Here we show that RecJ nuclease alone can initiate nucleolytic resection of DNA with 5′-ssDNA overhangs, and that RecQ helicase can initiate resection of DNA with blunt-ends or 3′-ssDNA overhangs by DNA unwinding. We establish that in addition to its well-known ssDNA exonuclease activity, RecJ can display dsDNA exonuclease activity, degrading 100–200 nucleotides of the strand terminating with a 5′-ssDNA overhang. The dsDNA product, with a 3′-ssDNA overhang, is an optimal substrate for RecQ, which unwinds this intermediate to reveal the complementary DNA strand with a 5′-end that is degraded iteratively by RecJ. On the other hand, RecJ cannot resect duplex DNA that is either blunt-ended or terminated with 3′-ssDNA; however, such DNA is unwound by RecQ to create ssDNA for RecJ exonuclease. RecJ requires interaction with SSB for exonucleolytic degradation of ssDNA but not dsDNA. Thus, complementary action by RecJ and RecQ permits initiation of recombinational repair from all dsDNA ends: 5′-overhangs, blunt, or 3′-overhangs. Such helicase–nuclease coordination is a common mechanism underlying resection in all organisms.

Homologous recombination is a relatively error-free mechanism to repair double-stranded DNA (dsDNA) breaks (DSBs) and single-stranded DNA (ssDNA) gaps, which are produced by UV light, γ-irradiation, and chemical mutagens (1). In wild-type Escherichia coli, the labor of recombinational repair is divided between the RecBCD and RecF pathways of recombination, which are responsible for the repair of DSBs and ssDNA gaps, respectively (2–5). However, the proteins of the RecF pathway are capable of DSB repair, as well as ssDNA gap repair: in recBC mutant cells containing the suppressor mutations, sbcB andsbcC (suppressors of recBC), the proteins of the RecF pathways provide the needed recombinational DNA repair functions (2, 6).

Consistent with this, in the absence of either repair or nascent DNA degradation, the recovery of replication is delayed, and both survival and recovery become dependent on translesion synthesis by DNA polymerase V (12, 13). 


RecF, 
RecO, and 
RecR


limit the RecJ/RecQ-mediated degradation and enhance the formation of RecA filaments at the arrested region (11, 14, 60, 64). Biochemical characterizations suggest that the RecA filament formed in the presence of RecFOR is capable of promoting branch migration at the fork in a manner that could promote regression away from the lesion and subsequently reset the 3′ end of the fork once the impediment has been removed or overcome (47, 60, 64, 69). In vivo, cells lacking any one of these gene products fail to resume DNA synthesis, and the DNA at the replication fork is extensively degraded (11, 14, 15).

Several lines of evidence suggest that Rep and the primosome also participate in restoring replication following arrest at a UV-induced lesion, either through direct resumption of the arrested replisome or de novo initiation of a replisome downstream of the arrest site. Both priA and rep contribute to the DNA synthesis that occurs during recombinational processes (26, 32, 41, 52, 65). Although no single gene by itself is essential for viability, double mutants in priA and priC or priA and rep are lethal, and both priA and rep mutants are hypersensitive to DNA damage (53). It has also been widely postulated that frequent replication disruptions by endogenous DNA damage in vivo account for the poor growth and low viability of priA and rep mutants (8, 45, 57). In addition, one study has reported a delayed recovery of DNA synthesis in PriA mutants following low doses of UV light (51). In vitro, the addition of PriA and PriB, PriA and PriC, or PriC and Rep allows DNA synthesis to occur at synthetic DNA fork structures in the presence of the other core replication proteins (25, 26). However, the role of PriA, PriB, PriC, and Rep in the progressive steps of resection, processing, or resumption following replication arrest at UV-induced DNA damage has not been directly examined in vivo. Here, we characterize the molecular events that occur during the progressive steps associated with the recovery of replication in UV-irradiated cultures of mutants lacking each of these gene products.


Two models that have been proposed for how the primosome and Rep helicase participate in restoring an active replisome following arrest by DNA damage are summarized in Fig. 6. Both models propose late functions for the primosome and Rep helicase but differ in the mechanism by which they promote replication recovery. The first model proposes that following arrest, the replisome and helicase are disrupted. Combinations of either PriA, PriB, or Rep with PriC participate in the displacement of the nascent lagging strand. These proteins then facilitate a transient loading of the helicase and primase complex on the leading-strand template, which serves as a primer, allowing a replisome to reinitiate downstream from the site of arrest (Fig. 6A). This model arose from the observation that, in vitro, the helicase activity of either PriA or Rep was capable of displacing the strands of a synthetic replication fork structure. In the presence of the helicase loader, DnaC, this is sufficient for the helicase and primase to prime the resulting single-stranded regions that are generated on the leading- and lagging-strand templates in vitro (22, 27).

The second model proposes that the primosome's primary contribution relates to enhancing the replisome's stability or priming efficiency during basal replication. Following arrest by UV-induced DNA damage, the helicase remains associated with the lagging strand, but other components of the holoenzyme may be displaced or disrupted. RecQ and RecJ contribute to the displacement and partial degradation of the nascent lagging strand, while the RecFOR proteins, together with RecA, process the fork DNA such that the lesion can either be repaired or bypassed. Once the block to replication has been overcome, the replisome can resume from the original arrest site. However, reestablishing an efficient replisome requires the primosome protein PriA and, to a lesser extent, PriB and PriC to coordinate the helicase/primase complex with the progressing replisome. The Rep helicase may also contribute to this reaction by helping to clear the region of other protein factors, such as recombination proteins, repair enzymes, or translesion polymerases, that may impair or compete with the replisome's ability to bind its forked substrate (Fig. 6B).





FIG 6
Two models for primosome and Rep function following disruption by DNA damage. 
(A) A model proposing that PriA and Rep function specifically to reinitiate DNA synthesis following disruption events. 
(i) Following the disruption of the replication machinery (grayed circles) by DNA damage (∧), 
(ii) PriA or Rep functions in a reaction to transiently load DnaB and DnaG to prime the leading strand and then 
(iii) stably load DnaB and DnaG on the lagging strand (22, 27). 
(iv) The leading-strand primer allows for the de novo formation of an active replisome downstream from the site of disruption. 
(B) A model in which PriA and Rep are required by the replisome to maintain efficient replication. 
(i) Following disruption by DNA damage, the recovery of DNA synthesis requires that the lesion is either repaired 
(ii) or bypassed 
(iii) by translesion synthesis (not shown), as found in previous studies (13). 
(iv) Since PriA and Rep are needed to maintain replication in the absence of damage, PriA and Rep would also be required for an active replisome to be maintained once the replisome is reestablished and DNA synthesis resumes.


Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT 1

Replication restart primosome is a complex dynamic system that is essential for bacterial survival. This system uses various proteins to reinitiate chromosomal DNA replication to maintain genetic integrity after DNA damage. The replication restart primosome in Escherichia coli is composed of PriA helicase, PriB, PriC, DnaT, DnaC, DnaB helicase, and DnaG primase. The assembly of the protein complexes within the forked DNA responsible for reloading the replicative DnaB helicase anywhere on the chromosome for genome duplication requires the coordination of transient biomolecular interactions. Over the last decade, investigations on the structure and mechanism of these nucleoproteins have provided considerable insight into primosome assembly. In this review, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.

A hand-off mechanism for PriA-directed primosome assembly [15] has been proposed (Figure 2), whereby 


(i) PriA recognizes and binds to a replication fork; 
(ii) PriB joins PriA to form a PriA-PriB-DNA ternary complex; 
(iii) DnaT participates in this nucleocomplex to form a triprotein complex, in which PriB is released from ssDNA due to recruitment of DnaT; 
(iv) the PriA-PriB-DnaT-DNA quaternary complex loads the DnaB/C complex; 
(v) DnaB is loaded on the lagging strand template. Genetic analyses suggest that these primosomal proteins are essential replication proteins for bacterial cell growth [12, 16–21].


These proteins are required for reinitiating chromosomal DNA replication in bacteria; thus, blocking their activities would be detrimental to bacterial survival [22, 23]. Several primosomal proteins, such as PriA, PriB, PriC, and DnaT, are not found in humans;



Figure 2: A hand-off mechanism for the replication restart primosome assembly. The proposed assembly mechanism is as follows. 
(i) PriA recognizes and binds to a replication fork, 
(ii) PriB joins PriA to form a PriA-PriB-DNA ternary complex, 
(iii) DnaT participates in this nucleocomplex to form a triprotein complex, in which PriB is released from ssDNA due to recruitment of DnaT, 
(iv) the PriA-PriB-DnaT-DNA quaternary complex loads the DnaB/C complex, and 
(v) DnaB is loaded on the lagging strand template.

Over the past 10 years, considerable progress has been made in the structural mechanisms of the replication restart primosome assembly. The structural information is a prerequisite for formulating any model of the assembly mechanism of the primosome (Table 1). In the following sections, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.


PriA Helicase

PriA helicase and SSB interact physically and functionally 2

PriA helicase is the major DNA replication restart initiator in Escherichia coli and acts to reload the replicative helicase DnaB back onto the chromosome at repaired replication forks and D-loops formed by recombination. We have discovered that PriA-catalysed unwinding of branched DNA substrates is stimulated specifically by contact with the single-strand DNA binding protein of E.coli, SSB. This stimulation requires binding of SSB to the initial DNA substrate and is effected via a physical interaction between PriA and the C-terminus of SSB. Stimulation of PriA by the SSB C-terminus may act to ensure that efficient PriA-catalysed reloading of DnaB occurs only onto the lagging strand template of repaired forks and D-loops.

INTRODUCTION

Genome duplication presents a formidable enzymatic challenge, requiring the high fidelity replication of millions of bases of DNA. Moreover, DNA replication occurs in a complex environment. The template is an inherently unstable polymer subject to a constant barrage of chemical insults (1), whilst conflicts between replication and other essential processes such as transcription are unavoidable (2–4). As a result, replication forks may stall frequently and require some form of repair to allow completion of chromosomal duplication (5,6). Failure to solve these replicative problems comes at a high price, with the consequences being genome instability, cell death and, in higher organisms, cancer. Prokaryotic studies have highlighted the central role played by recombination enzymes in fork repair (7). Damaged replication forks appear to have two fates in Escherichia coli. First, they may be processed so that the original blocking lesion is removed or bypassed, and replication resumed once the replicative machinery has been reloaded back onto the DNA fork structure (3,8,9). Second, stalled replication forks may break to leave one intact duplex and a free duplex DNA end (9–11). Recombination of the free duplex end with the intact sister duplex creates a D-loop onto which the replication machinery can be reloaded (12).
In both proposed replication repair pathways, the final stage of repair requires the restart of DNA replication. The key to initiation of DNA replication is loading of the replicative helicase DnaB onto ssDNA. DnaB catalyses unwinding of the parental DNA strands (13) and facilitates assembly of the remaining components of the replisome (14). Loading of DnaB during initiation of chromosomal duplication in E.coli is catalysed by DnaA in a tightly regulated manner at the start of the cell cycle and at a specific locus within the chromosome, oriC (15). In contrast, replication fork repair and hence reloading of DnaB may be needed away from oriC at any point within the chromosome and at any stage during chromosomal duplication. The potentially catastrophic effects of uncontrolled initiation of chromosomal duplication on genome stability suggests that replication restart must be regulated as tightly as DnaA-directed replication initiation at oriC. This implies reloading of DnaB must occur only on ssDNA at repaired forks or D-loops rather than onto other regions of ssDNA, such as those created by blocks to lagging strand synthesis (16,17). Thus an alternative replication initiator protein, PriA helicase, is utilized during replication restart to reload DnaB back onto the chromosome (18).
The requirement to reload DnaB only onto repaired forks and D-loops is thought to be reflected in the preferential binding of PriA to branched DNA structures in vitro (19,20). At such structures, PriA displays two activities. PriA facilitates loading of DnaB onto the lagging strand template via a complex series of protein–protein interactions involving PriB, PriC and DnaT (21–24). However, DnaB can bind only to ssDNA (13). Thus the second enzymatic function of PriA, a 3′ to 5′ DNA helicase activity (25), acts to unwind any lagging strand DNA present at the fork to generate a ssDNA binding site for DnaB (26). The importance of PriA-directed replication restart is underlined by the decrease in viability, defective homologous recombination and extreme sensitivity to exposure to DNA damaging agents exhibited by priA null strains (27–29). There exists also an alternative pathway of replication restart that is not dependent on PriA but on Rep helicase (24). Although rep mutants do not show the extreme phenotypes displayed by priA defective cells (30), rep and priA mutations are synthetically lethal (9,24). This suggests that Rep helicase may provide an accessory replication repair function. However, molecular details of the interplay between PriA- and Rep-dependent replication repair pathways remain unknown.
Here we show that SSB stimulates PriA-catalysed unwinding of branched DNA substrates. This stimulation requires binding of SSB to the initial DNA substrate and contact between PriA and the C-terminus of SSB. In contrast, neither a physical nor a functional interaction was detected between SSB and Rep helicase. A mutation within the C-terminus of SSB impairs interaction with PriA in vitro, and correlates with the DNA repair and recombination defects seen in strains carrying this ssb mutation. Contact between SSB and PriA may therefore play a critical role in coordinating reloading of the replisome at repaired forks and D-loops.

Crystal structure of DnaT 84–153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode 3

ABSTRACT
DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriAdependent primosome assembly is still a black box. Here, we report the 2.83 A DnaT ˚ 84–153-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy
results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.















1) http://www.hindawi.com/journals/bmri/2014/195162/
2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC535688/
3) http://nar.oxfordjournals.org/content/early/2014/07/22/nar.gku633.full.pdf
4) http://jb.asm.org/content/194/15/3977.full
5) http://web.pdx.edu/~justc/papers/recJrecQpaper.pdf
6) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260596/
7) http://europepmc.org/articles/PMC3710785
8 ) https://en.wikipedia.org/wiki/RecQ_helicase
9) http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Baxter/BLMgene.html
10) http://www.ebi.ac.uk/interpro/downloads/protein/prot_foc_12_06.pdf
11) http://www.nature.com/nrc/journal/v3/n3/full/nrc1012.html
12) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1373692/

Bacteria Are Champion Proofreaders  1

A team of Australian biochemists has examined the structure of just one of the “proofreading enzymes” in E. coli bacteria in unprecedented detail, and formulated a hypothesis for how it works.  That it does work, and works extremely well, is described in the introduction to their paper published in the April issue of Structure:

Fidelity of DNA replication is determined by three processes: base selection by a DNA polymerase, editing of polymerase errors by an associated 3'-5' exonuclease, and postreplicative mismatch repair.  In Escherichia coli, these processes contribute to duplication of the genome by the replicative DNA polymerase III (Pol III) holoenzyme with error frequency ~10^-10 per base pair replicated.

In other words, with its proofreading machinery, the bacterium makes a error once in 10 billion DNA letters.  Assuming 2000 letters on a page of single-spaced printed text, that would be roughly equivalent to one typo in about five million pages.

This high degree of fidelity is necessary; without it, errors would accumulate rapidly, causing the complete breakdown of the genome in a phenomenon called error catastrophe.  How did a lowly bacterium achieve such accuracy?  The paper mentions evolution three times, but never explains how such a system evolved; it just assumes that it did, and notes that the equipment is “highly conserved” (unchanged throughout living things).
In the new film on intelligent design, Unlocking the Mystery of Life, Dr. Michael Behe describes how he came to doubt Darwinian evolution.  He went through college never hearing how Darwinian evolution could explain the cell, but just was told that it did explain it, and assumed it to be true.  When he heard some convincing scientific arguments against Darwinism, he said he became somewhat angry, because he felt he had been led down the primrose path– he had gone through a graduate program, obtained a doctorate in biochemistry and became a university faculty member, and he had never heard of these things.  The arguments made him very interested in the subject of evolution, and he since concluded that Darwinian mechanisms are not sufficient to explain the complexity of life; they are “very inadequate,” in his opinion.
High-fidelity proofreading is just one of thousands of evidences that undirected natural processes are insufficient to produce the wonders in living cells.  That is part of what makes the case for intelligent design so convincing to more and more scientists and science teachers.  Too bad the authors of this otherwise wonderful scientific paper are still on the primrose path.

1) http://creationsafaris.com/crev0402.htm

Did DNA replication evolve twice independently? 1

DNA replication is central to all extant cellular organisms. There are substantial functional similarities between the bacterial and the archaeal/eukaryotic replication machineries, including but not limited to defined origins, replication bidirectionality, RNA primers and leading and lagging strand synthesis. However, several core components of the bacterial replication machinery are unrelated or only distantly related to the functionally equivalent components of the archaeal/eukaryotic replication apparatus.
Consequently, the modern-type system for double-stranded DNA replication likely evolved independently in the bacterial and archaeal/eukaryotic lineages.

This should be one more reason to doubt of the endosymbiotic theory


1) http://nar.oxfordjournals.org/content/27/17/3389.full

Origin and Evolution of DNA and DNA Replication Machineries

Origin and Evolution of DNA Replication Mechanism

1

Viral DNA Replication Mechanisms

In contrast to cellular genomes, which are all made of double-stranded DNA, viral DNA genomes are very diverse; some viruses have circular or linear double-stranded DNA genomes, while others have circular single-stranded DNA genomes.11 Single-stranded DNA genomes are replicated via rolling circle replication with a double-stranded DNA intermediate, whereas double-stranded viral DNA genomes are replicated either via classical theta or Y-shaped replication (for circular and linear genomes, respectively), by rolling circle, or by linear strand displacement11 (for recent reviews on eukaryal viral DNA replication, see ref. 31). In addition, replication can be symmetric, with both strands replicated simultaneously, but also asymmetric (the two strand are replicated not simultaneously but one after the other) or semi-asymmetric (the initiation of DNA replication on one strand being delayed until the first one is already partly replicated) (fig. 1). Some viral replication mechanisms are also used by plasmids (rolling circle) and some plasmids encode DNA replication proteins homologous to viral ones (see below), suggesting that plasmids originated from ancient viruses that have lost their capsid genes.26
The initiation of viral DNA replication needs a specific viral encoded initiator protein that can be a site-specific endonuclease (rolling-circle replication) or a protein that trigger double-stranded unwinding. Interestingly, plasmid and viral endonucleases involved in rolling-circle replication are evolutionary related.32The minimal recruitment for DNA chain elongation is a DNA polymerase. In contrast to RNA polymerases, all DNA polymerases (viral or cellular) need a 3'OH primer to initiate strand synthesis. This primer can be a tRNA (for reverse transcriptases), or a short RNA, either produced by a classical RNA polymerase (also involved in transcription) or a DNA primase. This use of RNA to initiate DNA synthesis is also often considered as a relic of the RNA world.
Some primases have a strong DNA polymerase activity, suggesting that primases testify for the transition between RNA and DNA polymerases.33 The definition of a DNA polymerase is thus becoming less straightforward, as also demonstrated by the recent characterization of DNA polymerases of the Y family that are involved in DNA repair and synthesize very short patches of DNA (much like a primase)25,34 and by the discovery of structural similarities between eukaryal primase and DNA polymerases of the family X.35
As a consequence of the ancient metabolic pathway producing only 5' nucleotides, the strand moving in the 3' to 5' direction in symmetric or semi asymmetric replication has to be replicated backward in the form of short DNA pieces (Okazaki fragments) (fig. 3). These fragments are primed by DNA primase and later on assembled by a DNA ligase, after removal of the RNA primer by RnaseH or various exonuclease activities, sometimes associated to DNA polymerases. In some cases of asymmetric replication (Adenovirus, bacteriophage Φ29, mitochondrial linear plasmids), the DNA polymerases use a protein priming system to produce a free 3'OH for the DNA polymerase. All polymerases using this system belong to a subfamily of the DNA polymerase B family.25
Some DNA polymerases can perform strand displacement that is required for asymmetric DNA replication, while others, in order to improve the efficiency of this process associate with DNA helicases and/or single-stranded DNA binding proteins (ssb) to unwind the two DNA strands. The processivity of many viral DNA polymerases is further enhanced by specific processivity factors. In the case of T4, these include ring-shaped DNA clamps, and hand-shaped clamp-loader complexes that can open and close the ring-shaped DNA clamp around the DNA molecule.
In symmetric replication, the syntheses of the leading and lagging strands are coupled via an interaction between the primase and the helicase (fig. 4). In some bacteriophages (T7, P4) and eukaryal viruses (Herpes), this coupling is achieved by the fusion of the helicase and the primase activities into a single polypeptide.36,37 This is clearly a case of convergent evolution, since bacteriophages and Herpes primases belong to different protein families.38

Figure 4

Evolution of DNA replication mechanisms from the simple asymmetric mode to the symmetric mode (or vice versa). In the fully asymmetric mode (top) that occured in RNA and DNA viruses, one strand is replicated entirely before the initiation of replication of the displaced strand. The minimal requirement of this mechanism is a DNA polymerase and a priming system. Strand displacement can be made more efficient by the recruitment of processivity factors and a helicase. In the semi-asymmetric mode (middle) a DNA primase initiates replication on the displaced strand before termination of the replication of the first (leading) strand. In the symmetric mode (bottom) coupling between primase and helicase allows the displaced strand (now the lagging strand) to be replicated together with the leading strand.

The two DNA polymerases that replicate the lagging and the leading strands can be also physically linked. As a consequence, the lagging strand loops upon itself, and the two strands are replicated at once very rapidly, limiting the presence of single-stranded DNA to the fork vicinity. This is in striking contrast with asymmetric replication that requires complete denaturation of the two strands before replication of the lagging strand (fig. 4).
Some DNA viruses replicate their genome using only replication proteins encoded by their host (with the exception of initiator proteins). However, many large DNA viruses encode also several proteins involved in the elongation step of DNA replication. Some of them (e.g., T4-phages) have reached a high level of complexity in their DNA replication machinery, and consequently encode functional analogs for all proteins involved in cellular DNA replication (fig. 5).39

Figure 5

The universal replication fork for symmetric theta replication. Proteins with different activities are indicated with different colours and their usual names indicated for A= Archaea (Ae=euryarchaea, Ac=crenarchaea, B=Bacteria, E=Eukarya, and bacteriophage T4. Homologous proteins performing the same function are framed together. Letters in brackets indicate DNA polymerase families. The looping of the lagging strand, which allows concomitant replication of the leading and the lagging strand by a single replicase factory, is supported by experimental evidence for Bacteria and T4 phage. For an exhaustive analysis of the phylogenetic relationships between different cellular replication proteins see reference 48.


Considering that replication of double-strand RNA viruses is completely asymmetric, it is likely that DNA replication first occurred via the asymmetric mode and evolved toward fully symmetric theta mode via the semi-asymmetric mode (fig. 3). If viruses recruited their DNA replication mechanisms from the cells, as proposed in the “escaped theory” for viral origin, this means either that viruses originated from early DNA cells that have not yet reached the stage of the symmetric mode of replication, or that this mode has been modified in many viruses to produce simpler systems. The latter possibility cannot be excluded, since there is some plasticity in the evolution of DNA replication mechanisms, and this evolution is not necessarily unidirectional (fig. 4). For example, the replication of bacterial chromosome during conjugation can be changed from the symmetric theta mode to the asymmetric rolling-circle mode upon the integration of a conjugative plasmid.11
On the contrary, if DNA originated in viruses (7), one can even imagine that several DNA replication systems emerged and evolved independently from different lineages of RNA viruses. This hypothesis thus allows for a long period of DNA replication evolution purely in the viral world (fig. 2). This would nicely explain the existence of different version of functionally analogs but nonhomologous DNA replication proteins. The diversity of viral replication proteins can be exemplified by those of Pox virus, Herpes viruses or T4, that are completely different from each others, and are no more related to the archaeal/bacterial systems (in term of protein similarities) than these systems are related between each others.31,36,37,39 Recent sequencing of the 280 kbp bacteriophage phiKZ of Pseudomonas aeruginosa failed to identify virus-encoded DNA replication-associated proteins, suggesting that they may be strongly divergent from known homologous proteins.40 Finally, it is noteworthy that several families of proteins involved in DNA replication also appears restricted to the virus world, such as helicase of the superfamily III,41 the Herpes primases,38 or protein-primed DNA polymerases of the B family.25 Some linear mitochondrial plasmids also encode the latter enzyme, again suggesting a connection between viruses and plasmids. The recent discovery of a completely new family of DNA polymerase/primase encoded by the archaeal plasmid pRN2 once more emphasizes the potential of viruses and plasmids as source of novel DNA replication proteins.78 It is difficult to understand the existence of all these viral and/or plasmid specific DNA replication proteins in the framework of the “escaped theory” for the origin of viruses. On the contrary, in the viral origin hypothesis, these enzymes have simply originated in viruses and were never been transferred to the cells.

Cellular DNA Replication: Two Independent Inventions

In all cells, DNA replication occurs by a symmetric (theta) mode of replication. The proteins involved and their mechanisms of action have been analyzed in much details during these last decades in several bacterial and eukaryal model systems.11,31,43 The basic principles of DNA replication are very similar inBacteria and Eukarya, and probably in Archaea as well (fig. 5).44,45 For the initiation step, initiator proteins recognize specific DNA sequences at replication origin(s). A loading factor then brings the replication helicase to the initiation complex to start the assembly of the replisome. The movement of the replication forks involves the concerted action of primases, DNA helicases, ssb proteins, and at least two processive DNA polymerases (with clamp and clamp loading factors) to couple replication of the leading and lagging strands, allowing the efficient replication of large cellular genomes. In turn, type II DNA topoisomerases became essential to solve the topological problems due to the unwinding of the double-helix in such large molecules, counteracting the production of positive superturns ahead of the forks and allowing separation of daughter molecules. This mechanism of DNA replication strikingly resembles those of some large DNA bacteriophages, such as T4 (fig. 5).
Originally, the striking similarity between the enzymatic activities involved in bacterial and eukaryal DNA replication suggested that they originated from a common ancestral DNA replication mechanism already present in LUCA (in the nomenclature of the evolutionists, the bacterial and eukaryal DNA replication proteins were supposed to be orthologues, i.e., to have evolved in parallel to speciation from a common ancestor). In that case, bacterial, eukaryal and archaeal DNA replication proteins performing analogous function should be orthologous. However, comparative genomic analyses have shown that this is not the case (fig. 5).46-48 On the contrary, several critical DNA replication proteins identified in Bacteria by genetic and in vitro analyses have no homolog in Archaea or Eukarya, whereas others have only very distantly related homologues that are probably not orthologues. Similarly, most DNA replication proteins previously identified in Eukarya turned out to have readily detectable homologues only in Archaea.
The similarity between DNA replication proteins in Archaea and Eukarya is especially remarkable. It cannot be due to functional convergence since they have somewhat different modes of replication (unique origin and high-speed in Archaea, multiple origin and low speed in Eukarya),49 whereas Archaea andBacteria have dissimilar replication proteins but identical replication mode (unique origin, high speed, hot spot of recombination at the replication terminus, and major genomic recombination events occurring between bi-directional replication forks.)49-50 The high level of similarities between the archaeal and eukaryal DNA replication proteins also cannot be explained by similar chromatin structure (as suggested by Cavalier-Smith),51 since most archaeal proteins involved in DNA replication are similar in the two archaeal phyla the Crenarchaeota and the Euryarchaeota, whereas the presence of eukaryal-like histones is restricted to the Euryarchaeota.
Five alternative hypotheses have been proposed to explain the evolutionary gap between the bacterial and the eukaryal/archaeal replication systems (fig. 6).



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[*]the replication proteins of Bacteria and Archaea/Eukarya are actually orthologues, but they have diverged to such an extent that their homology cannot be detected anymore at the sequence level.46


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[*]two different replication systems were present in the LUCA; one was retained in Bacteria, the other in Archaea/Eukarya.46

[*]LUCA had an RNA genome, and DNA and DNA replication were invented twice independently, once in Bacteria and once in the ancestral lineage common to Archaea and the Eukarya.47-48

[*]The ancestral replication mechanism present in LUCA has been displaced either in Bacteria or in Archaea/Eukarya by a new one, corresponding to a nonorthologous displacement.46,52 More specifically, it has been suggested that the bacterial replication system, or part of the eukaryal one, are of viral origin.52 - 53

[*]Both bacterial, archaeal and eukaryal replication mechanisms are of viral origin and have been transferred to cells independently.7


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Figure 6

The different hypotheses for the origin and evolution of DNA and DNA replication mechanisms. A=Archaea, B=Bacteria, E=Eukarya. The universal trees of life are unrooted, except in the case of hypotheses 1, 3 and 5, which favor the bacterial rooting.3-5 White circle: the archaeal/eukaryal DNA replication proteins: black circle: the bacterial DNA replication protein). The large gray circle represents LUCA. See the text for explanations.

The different hypotheses for the origin and evolution of DNA and DNA replication mechanisms. A=Archaea, B=Bacteria, E=Eukarya. The universal trees of life are unrooted, except in the case of hypotheses 1, 3 and 5, which favor the bacterial rooting.- White (more...)
The hypotheses 4 and 5 can be combined, if a first transfer from viruses to cells occurred before LUCA, and a second one displaced this ancestral cellular mechanism later on.
In addition several authors have proposed that the eukaryal nucleus itself originated from a large DNA virus (possibly an archaeal virus) that could be related to Poxviruses.54-55
The first hypothesis (the hidden orthology) can be clearly ruled out, since the bacterial and the archaeal/eukaryal versions of the two central players in the elongation step of DNA replication, the replicative polymerases and the primases, belong to different protein families.25,35,48 In the case of primases, structural analyses have shown that the bacterial and the eukaryal/archaeal versions are completely unrelated, the latter being member of the DNA polymerase X family.35 In the case of the replicase, the structure of the bacterial one (PolC/DnaE) has not yet been solved, but in-depth sequence analysis failed to detect any similarity with the superfamily of RNA polymerases, reverse transcriptase and DNA polymerases of the A and B families.48
In other cases (the replicative helicase, the single-stranded DNA binding proteins, the initiator proteins), comparative structural analyses and/or PSI-BLAST searches have shown that the bacterial and eukaryal/archaeal proteins belong to same superfamilies, since they share homologous domains. However, they are clearly not orthologues, since they belong to different families. For example, in the case the initiator protein (DnaA in Bacteria, Cdc6/Orc1 inArchaea and Eukarya) the bacterial and archaeal proteins share a common ATPase module of the same family (AAA+), but these modules are associated to different modules that are probably involved in DNA binding.57
The bacterial and archaeal/eukaryal versions of many DNA replication proteins have thus been certainly invented independently, probably by recruitment and modification of proteins previously involved in RNA replication and/or RNA gene regulation. However, a few DNA replication proteins (the clamp, the clamp loader, DNA ligase) could be orthologous in the three domains of life since they share sequence similarities that can be detected by elaborated PSI-BLAST analyses or structural similarity with unique fold and fold arrangement.48Furthermore, they are more similar to each other's, from one domain to another, than to any other proteins. We should thus explain why different replication systems that have emerged independently use some homologous accessory proteins. It is possible that these proteins originated late in the history of DNA replication and were independently recruited by evolving DNA replication systems. Alternatively, they might have predated DNA replication itself and were independently used by different emerging systems.
In order to better understand the evolution of the DNA replication apparatus, it would be necessary to determine with some confidence when and where the independent inventions of the bacterial and the eukaryal/archaeal versions of nonorthologous DNA replication mechanisms occurred (either before or after LUCA, either in cells or in viruses?). We will discuss now several specific points of the above hypotheses (except hypothesis 1 that we have ruled out) in an attempt to answer some of these questions.

1) http://www.ncbi.nlm.nih.gov/books/NBK6360/

Under this replication-centered perspective, the emergence of complexity is an enigma: Why are there numerous life forms that are far more complex than the minimal, simplest device for replication? We cannot know “for sure” what these minimally complex devices are, but there are excellent candidates —namely, the simplest autotrophic bacteria and archaea, such as Pelagibacter ubique or Prochlorococcus sp. These organisms get by with about 1,300 genes without using any organic molecules, and generally without any dependence on other life forms. Incidentally, these are also the most “successful” organisms on Earth. They have the largest populations that have evolved under the strongest selection pressure—and consequently have the most “streamlined” genomes. A complete biosphere consisting of such highly effective unicellular organisms is easily imaginable; indeed, the Earth biota prior to the emergence of eukaryotes (that is, probably for the 2 billion years of the evolution of life or so) must have resembled this image much closer than today’s biosphere (although more complex prokaryotes certainly existed even at that time).

So why complex organisms?


One answer that probably appeared most intuitive to biologists and to everyone else interested in evolution over the centuries is that the more complex organisms are also the more fit. This view is demonstrably false. Indeed, to accentuate the paradox of complexity, the general rule is the opposite: The more complex a life form is, the smaller effective population size it has, and so the less successful it is, under the only sensible definition of evolutionary success. This pattern immediately suggests that the answer to the puzzle of complexity emergence could be startlingly simple: Just turn this trend around and posit that the smaller the effective population size, the weaker the selection intensity, hence the greater the chance of non-adaptive evolution of complexity. This is indeed the essence of the population-genetic non-adaptive concept that Lynch propounded.

from the book: The Logic of Chance: The Nature and Origin of Biological Evolution By Eugene V. Koonin page 266

Tiny Bacteria's Big Challenge to Darwin 1

Bacteria (prokaryotes) are found everywhere and are a critical foundation of the earth's ecosystem. The prokaryotes are designed to be saprotrophs or "decomposers," breaking down wastes and organic material so that chemical components such as nitrogen can be recycled.
Evolutionary theory states that some ancient prokaryotes ("simple" forms) evolved into eukaryotes ("complex" forms). Eukaryotes are cells with a membranebound nucleus and DNA structured into linear chromosomes, versus the circular chromosomes in bacteria. However, the Creator has designed bacteria with some amazing properties that should cause one to be openly skeptical of Darwinian claims regarding bacteria's origin and alleged evolution over time into completely different life forms. (For example, see my article "Just How Simple Are Bacteria?"1)
Secular scientists have no credible idea how the DNA molecule may have evolved from non-life--especially without the aid of proteins (which must be coded by DNA 2) or the critical DNA repair system.3
The genetic material (DNA) in most bacteria is found as a single circular chromosome in an area called the nucleoid region and contains some 4.7 million base pairs. Stretched out, this DNA molecule would be about 1,000 times longer than the bacterium itself. The bacterial chromosome, though chemically identical, is structured unlike linear chromosomes of eukaryotic cells that make up people (46 chromosomes), plants (e.g., corn, 20 chromosomes) and animals (e.g., fruit fly, 8 chromosomes).
All cells (except mature red blood cells) must duplicate their genetic material for the next generation. The process of DNA duplication in bacteria, called replication, into two exact copies--one for each new daughter cell--is quite complex. This involves an origin site on the circular molecule (called oriC) where replication begins. Then, bidirectional replication of the two strands at identical speeds is carried out with precision. As you can see, this is hardly simple and involves many enzymes, including topoisomerases. These large molecules are designed with the important job of relaxing and uncoiling the DNA. Some anti-cancer drugs work by interfering with topoisomerases in targeted cancer cells.
As impossible as it would have been for such a process to have evolved through time, chance, and random genetic mistakes, three evolutionists ask if DNA replication could have evolved twice independently!4
Replication difficulties aside, fitting the convoluted mass of DNA in the confines of a tiny bacterium requires an amazing process called supercoiling. The Creator has designed enzymes that rapidly and efficiently twist the bacterial DNA upon itself. For example, Type II topoisomerases (DNA gyrase that produces negatively supercoiled DNA by cutting it) maintain a precise, steady-state degree of supercoiling. Fully supercoiled, the chromosome is about 1 μm (a micrometer, 1 millionth of a meter) in diameter, while its relaxed configuration is approximately 430 μm.
Far from supercoiling just being an efficient manner in which the bacterium stores its DNA, researchers are discovering that "supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell." 5 A second messenger (e.g., cyclic AMP) is an intermediary compound that can alter fundamental patterns of gene (DNA) expression.
So, not only must the DNA of bacteria replicate error-free at an amazing rate (30,000 "letters" per minute), but it must also be compacted to fit inside an impossibly small space. During replication, certain genes must also be immediately available for necessary bacterial functions, some actually being expressed by their sensitivity to supercoiling--which in turn is stimulated by environmental changes. And this is just the "simple" bacterium. As we say in creation science, "If it's living--it's complex!"


References


[1]Sherwin, F. 2001. Just How Simple Are Bacteria? Acts & Facts. 30 (2).


[2]See Sherwin, F. 2002. The Egg/Chicken Conundrum. Acts & Facts. 31 (5).


[3]See Sherwin, F. 2004. Mending Mistakes--The Amazing Ability of Repair. Acts & Facts. 33 (6).


[4]Leipe, D.D., L. Aravind, and E.V. Koonin. 1999. Did DNA replication evolve twice independently? Nucleic Acids Research. 27 (17): 3389-3401.


[5]Peter, B.J., et al. 2004. Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli. Genome Biology. 5 (11): R87.

1) http://www.icr.org/article/3942

DNA REPLICATION

There comes a time in the life of every cell when it turns to thoughts of division. One major consideration in cell division is ensuring that the genetic information be copied and handed down uncorrupted; a great deal of effort is invested in that task. In 1957 Arthur Komberg demonstrated that a certain enzyme could polymerize the activated forms of deoxynucleotides into a new DNA molecule that was an exact copy of whatever «template» DNA Komberg threw into the reaction mixture. He called the enzyme DNA polymerase I (Pol I). The scientific community was ecstatic about the find. Over the years, however, it has been shown that Pol I's primary role is not to synthesize DNA during cell division; rather, it is to repair DNA that has been damaged by exposure to ultraviolet light, chemical mu-THE CHEMISTRY OF LIFE 293 tagens, or other environmental insults. Two other DNA polymerases, Pol II and Pol III, were later discovered. The role of Pol II remains murky: mutant cells lacking the enzyme exhibit no observable defects, Pol III has been identified as the major enzyme involved in DNA replication in prokaryotes. DNA polymerase III is actually a complex of seven different sub-units, ranging in length from about 300 to about 1,100 amino acid residues. Only one of the subunits does the actual chemical joining of nucleotides; the other subunits are involved in critical accessory functions. For instance, the polymerizing subunit tends to fall off the template DNA after joining only ten to fifteen nucleotides. If this happened in the cell the polymerase would have to hop back on hundreds of thousands of times before replication was complete, slowing replication enormously. However, the complete Pol III—with all seven sub-units— does not fall off until the entire template DNA (which can be more than a million base pairs long) is copied. In addition to a polymerizing activity Pol III possesses, ironically, a 3'5' nuclease activity. This means that it can degrade polymerized DNA into free nucleotides, starting at a free 3' end and working back toward the 5' end. Now, why would a polymerase also degrade DNA? It turns out that the nuclease activity of Pol III is very important in ensuring the accuracy of the copying procedure. Suppose that the wrong nucleotide became incorporated into the growing DNA chain. Pol Ill's nuclease function allows it to step back and remove the incorrect, mis-paired nucleotide. Correctly paired nucleotides are resistant to the nuclease activity. This activity is called «proofreading»; without it, thousands of times more errors would creep in when DNA was copied. DNA replication begins at a certain DNA sequence, known appropriately as an «origin of replication,» and proceeds in both directions at once along the parent DNA. The first task to be tackled during replication, as for transcription, is the separation of the two parent DNA strands. This is the job of the DnaA protein. After the strands are separated two other proteins, called DnaB and DnaC, bind to the single strands. Two more proteins are recruited to the growing «bubble» of open DNA: single strand binding protein (SSB), which keeps the two parent DNA strands separated while the DNA is copied; and gyrase, which unknots the tangles that occur as the complex plows through double stranded DNA.

WHAT DOES THE BOX TELL US?

At this point DNA polymerase can begin synthesis. But several problems arise. DNA polymerase cannot start synthesizing by joining two nucleotides the same way that RNA polymerase starts transcription; the DNA enzyme can only add nucleotides to the end of a preexisting polynucleotide. Thus the cell employs another enzyme to make a short stretch of RNA on the exposed DNA template. This enzyme can begin RNA synthesis from two nucleotides. Once the RNA chain has gotten to be about ten nucleotides long, the DNA polymerase can then use the RNA as a «primer,» adding deoxynucleotides to its end. The second problem occurs as the replication «fork» opens up. The synthesis of one strand of new DNA can proceed without difficulty; this is the strand that the polymerase makes as it reads the template in a 3'5' direction, making a new strand in a 5'3' orientation, as all polymerases do. But how to synthesize the second strand? If done directly, the polymerase would have to read the template in a 5'3' direction and thus synthesize the new strand in a 3'5' direction. Although there is no theoretical reason why this could not occur, no known polymerase synthesizes in a 3'5' direction. Instead, after a stretch of DNA has been opened up, an RNA primer is made near the fork and DNA synthesis proceeds backward, away from the replication fork, in a 5'3' direction.  Further synthesis on this «lagging» strand must wait until the replication fork opens up another stretch of DNA; another RNA primer must then be made, and DNA synthesis proceeds backward toward the previously synthesized fragment. The RNA primers must then be removed, the gaps filled in with DNA, and the ends of the DNA pieces «stitched together.» This requires several more enzymes. The above description of prokaryotic DNA replication has been pieced together by the enormous efforts of a large number of laboratories. The replication of eukaryotic DNA appears to be much more complex, and therefore much less is known about it.

Behe, Darwin's black box, page 222

Prime-time looping

When the replication machinery copies DNA, it must unwind the double helix in one direction while synthesis of one of the strands proceeds in the other. Making transient DNA loops may solve this directional dilemma.

If you are a cell about to divide, you will first need to use a multi-protein machine called a replisome to simultaneously make copies of both strands of your chromosomal DNA so that one strand can be passed to each daughter cell. Replisomes have long been thought to couple synthesis of both DNA strands by forming a ‘trombone loop’ of DNA that expands and relaxes as synthesis takes place discontinuously on one of the strands. Two papers, one by Pandey et al.1 and another by Manosas et al. published in Nature Chemical Biology, show that a second type of loop, called the ‘priming loop’, is transiently produced in the replisome. The replisome faces special challenges as it makes new DNA at rates that can approach 1,000 nucleotides per second. Unlike the machines that make proteins and RNA, which work relatively sluggishly and in a linear fashion, the replisome must simultaneously copy two strands of DNA that are aligned in opposite directions (5ʹ to 3ʹ and 3ʹ to 5ʹ). Replisome chemistry obeys two rules. How did they arise with that cabability to " obey two rules " ?  The first is that a DNA polymerase (the component of the replisome that synthesizes new DNA from a template strand) can extend the newly formed DNA chain only in the 5ʹ to 3ʹ direction. This means that it can continuously copy only one of the two DNA strands, called the leading strand. The lagging strand must be made in shorter pieces that are joined together later. These pieces, or Okazaki fragments, are a few thousand bases in length and each is made every few seconds. The second rule is that a DNA polymerase cannot start a DNA chain — it can only extend a pre-existing DNA or RNA chain, called a primer. So all cells have a specialized enzyme, the primase, that makes the first RNA primer for each DNA chain. Question : Had the primase not have to arise at the same time together with the other proteins to make the primer? A new primer must therefore be made every few seconds to be used for Okazaki-fragment synthesis on the laggingstrand template. This single-stranded template DNA is produced by the helicase, a component of the replisome that, in bacteria, moves in a 5ʹ to 3ʹ direction to separate the two strands of the double helix (Fig. 1). 



Figure 1 | DNA replication by a minimal replisome. During DNA replication by the replisome components, the DNA strands are separated by the helicase enzyme and replicated by the leadingand lagging-strand DNA polymerases. As DNA can be copied only in the 5ʹ to 3ʹ direction, the polymerase continuously copies the leading strand, but the lagging strand is made in shorter pieces, or Okazaki fragments, that are joined together later. DNA synthesis begins by extending a nucleic-acid primer that is synthesized at priming sites by the primase enzyme.

And herein lies the problem — the primase needs to be associated with the helicase to function, but the primers on the lagging strand are made in the direction opposite to the movement of the helicase. Moreover, primer synthesis is relatively sluggish, taking about a second or so. There are three possible solutions to the replisome’s problem. One is for the whole replisome to pause while the primer on the lagging strand is made, then to resume its work; such pauses have been reported by the van Oijen group3 during primer synthesis by the bacterial virus (bacteriophage) T7 replisome (Fig. 2a).



Figure 2 | Three priming mechanisms. Interaction of the primase with the helicase is necessary for primer synthesis at a lagging-strand priming site. The primase makes primers in the opposite direction to helicase movement, leaving three ways by which the replisome might resolve this directional problem. a, The whole replisome can pause for primer synthesis; b, it can promptly release the primase; or c, as described for the first time by Pandey et al.1 and Manosas et al.2, the replisome can continue to move forward while the primase–helicase interaction persists. This produces a priming loop that eventually collapses into the lagging-strand trombone loop, probably on transfer of the primer to the lagging-strand polymerase.

The second solution is for the primase, once clamped onto the lagging-strand template by the helicase, to be promptly released to make its primer at leisure, as happens with the Escherichia coli replisome4 (Fig. 2b). The third solution is for the replisome to continue leading-strand synthesis while the helicase–primase complex takes its time to make the primer. The helicase continues to unwind DNA in the forward direction while the physically linked primase makes a primer in the opposite direction. This arrangement produces a transient single-stranded DNA loop in the lagging-strand template, termed the priming loop, which is subsequently released to become part of the trombone loop when the primer is passed to the lagging-strand polymerase (Fig. 2c). The new reports1,2 use elegant single-molecule experiments to provide the first direct experimental evidence for priming-loop formation by the bacteriophage T7 and T4 replisomes. Pandey et al.1 worked with the whole T7 replisome, which has an unusual structure in that its primase and helicase are part of the same protein, so primase release is impossible. The authors used short DNA templates that were already primed on the leading strand, with priming sites (DNA sequences required for primer synthesis) on the lagging strand. Although lagging-strand primer synthesis occurred about 50% of the time, synthesis of the leading strand showed no sign of pausing while a primer was made. Next, the authors1 employed a technique called fluorescence resonance energy transfer (FRET), which uses the interaction between fluorescent dyes as a read out of the proximity of molecules to each other. The dyes were arranged on the laggingstrand template so that they would come close enough together for FRET to occur if a priming loop were formed. FRET was observed only under conditions where, and about as often as, primers were made. The FRET data1 can be explained only by the formation of a priming loop on the lagging-strand template while leading-strand synthesis continues (Fig. 2c). In another single-molecule study, Manosas et al.2 studied the T4 replisome, in which the primase and helicase are separate proteins that interact during primer synthesis. They used an ingenious experimental design consisting of a double-stranded DNA hairpin structure that contains priming sites when in a singlestranded form. The DNA is attached to a magnetic bead that is stretched at a constant low force by a magnetic field. Videomicroscopy of the bead movement allows measurement of the length of the DNA. As the helicase converts the hairpin to single-stranded DNA, the DNA lengthens and then subsequently contracts as the hairpin reanneals behind it. The changes in DNA length allow measurement of the rate of helicase action in real time. Using this system, the authors2 showed that helicase–primase interaction and subsequent primer synthesis did not result in helicase pausing. Most of the time, reannealing of the hairpin was blocked by the persistence of a primase-bound primer, indicating that the primase had been released promptly by the helicase at the priming site (Fig. 2b). Less frequently, the rate of DNA lengthening decreased for about half a second, and then there was an immediate jump in length. This observation can be explained only by the formation and subsequent release of a priming loop (Fig. 2c). When the helicase and primase were artificially fused together as in the T7 replisome, priming-loop formation was markedly increased, and blocks to re annealing (by released primase-bound primer) were not observed. An unusual aspect of Pandey and colleagues’ work1 is the high efficiency of priming achieved by the T7 primase on their short templates. Priming sites are trinucleotides that occur frequently in single-stranded DNA templates. They are generally used inefficiently by the primase for primer synthesis, and it is thought that only a fraction of primers are functionally extended by the lagging-strand polymerase. These factors account for the relatively long (1–2 kilobases) Okazaki fragments. When studying lagging-strand priming during leading- strand synthesis by the T7 replisome on long templates, the van Oijen group3 clearly observed pauses coincident with primer synthesis. These occurred at relatively low frequency, consistent with the size of Okazaki fragments — but the authors’ single-molecule experimental set-up could not detect priming loops. Reconciliation of these observations3 with those of Pandey et al.1 is not straight forward, and may indicate that replisome pausing occurs during or soon after functional primer synthesis, while mechanisms involving primase dissociation and priming-loop formation ensure that the replisome is not unnecessarily slowed during more frequent, non-productive priming events.

Mechanisms and Controls of DNA Replication in Bacteria 1

Introduction

DNA is the polymeric molecule that contains all the genetic information in a cell. This genetic information encodes the instructions to make a copy of itself, for the cellular structure, for the operative cellular machinery and also contains the regulatory signals, which determine when parts of this machinery should be on or off. The operative machinery in turn, is responsible for the cells functions either metabolically or in interactions with the environment. Part of this cellular machinery devoted to DNA metabolism is responsible for DNA replication, DNA-repair and for the regulation of gene expression. In this chapter we will focus our discussion on the mechanisms and controls that conduct DNA replication in bacteria, including the components, functions and regulation of replication machinery. Most of our discourse will consider this biological process in Escherichia coli but when possible we will compare it to other bacterial models, mainly Bacillus subtilis and Caulobacter crescentus as examples of organisms with asymmetrical cell division. In order to maintain a bacterial population it is necessary that cells divide, but before the physical division of a daughter cell from its mother, it is necessary among other check points, that the DNA has been replicated accurately. This is done by the universal semiconservative replication process of DNA-strands, which generates two identical strand copies from their parent templates. To better understand this process it has been divided into three phases: initiation, elongation and termination of DNA replication. In each of these steps, multiple stable and transient interactions are involved and we have summarized them below.

The study of the cell-cycle in bacteria is usually divided into three stages: the period between cell-division (cell birth) and the initiation of chromosome replication, the period required to complete DNA replication (elongation of DNA) and, the final phase, which goes from the end of DNA replication until the completion of celldivision (Wang & Levine, 2009). Under the best growing-conditions, DNA replication starts immediately after cell division in most cells (Wang et al., 2005). Since replication of the chromosome takes more time than that the necessary for cell division under optimal culture conditions, such as E. coli growing in rich media, at 37ºC with good aeration, it can happen that more than one event of DNA replication can occur per cell cycle (Zakrzewska-Czerwinska et al., 2007). For the purposes of this work we shall divide the DNA replication process in bacteria into three steps: initiation, elongation and termination as follows.

3. Regulation of DNA replication 
The regulation of DNA replication is a vital cellular process. In a general view, DNA replication is controlled by a series of mechanisms that are centered on the control of cellular DnaA levels, its availability as a free protein and modulation of its activity by binding the small-molecule ligand ATP (Leonard & Grimwade, 2009); the other point of control is by modulating the accessibility of replisome components to the oriC region on the DNA. We discuss some aspects of these regulatory mechanisms below. 




3.1 Regulatory mechanisms of DNA replication in E. coli 

One of the main mechanisms associated with DNA replication is the so-called RIDA system (Regulatory Inactivation of DnaA). The elements of this system are the sliding-clamp of DNA polymerase III and Hda (Homologous to DnaA). This mechanism takes place when DnaA is activated by its binding to ATP. The accumulation of DnaA in this active form leads to the initiation of chromosomal replication since it facilitates its binding to the oriC on the DNA. DnaA reverts to its inactive form DnaA-ADP by hydrolysis of ATP (Katayama et al., 1998). Hda-ADP is the monomeric active form for promoting the hydrolysis of DNA-ATP, a process which is mediated by the slider-loader clamp (Su’etsugu et al., 2008). This inactivating regulation of DnaA is key for preventing the over-initiation of replicative events during the cell cycle (Katayama & Sekimizu, 1999). The free-living bacteria C. crescentus also presents this regulatory mechanism, as it has HdaA, a protein similar to the E. coli Hda. In C. crescentus HdaA also inactivates DnaA in a replication-coordinated manner, if DNA replication is successfully initiated then HdaA and the ┚-sliding clamp promote the hydrolysis of DnaA-ATP to DnaA-ADP and force DnaA to leaves the oriC (Collier & Shapiro, 2009). A conserved bacterial protein, YabA, has been found in B. subtilis and other Gram-positive bacteria where it acts as a repressor for initiation of DNA replication. This is achieved by forming a complex with DnaA and the ┚-sliding clamp independently of the DNA, a common activity shared between Hda and YabA (Mott & Berger, 2007). Thus the RIDA system is present in B. subtilis and is also the primary mechanism for regulation of DNA replication in this bacterium (Noirot-Gros et al., 2006). The formation of the oriC and DnaA complex is assisted by the protein DiaA, which forms homo-tetramers and binds various DnaA molecules, especially in the active form of DnaA-ATP but it can also stimulate the formation of the DnaA-ADP-oriC complex, this is an inactive complex for initiation of replication (Ishida et al., 2004).

Another mechanism that regulates the initiation of DNA replication is by controlling the availability of free DnaA to bind to DnaA boxes on the oriC . Here the role of the 1kb datA locus, which is localized near (downstream) from the oriC is important. The datA locus shows high affinity for DnaA, even more than the DnaA boxes on the oriC. Thus the datA region is able to bind over 300 DnaA molecules whereas oriC binds to 45 DnaA monomers (Kitagawa et al., 1998). The operability of this mechanism is facilitated by the fact that the oriC had only few DnaA boxes compared to the datA locus and by the close proximity of data in respect to oriC on the DNA molecule (Figure 6). 


Fig. 6. Mechanisms that regulate DNA replication in E. coli. A) The newly replicated DNA duplex is in a hemimethylated state. B) SeqA binds to the hemimethylated GATC sites immediately after they are replicated. C) RpoD activates the transcription of dam and Dam methylates GATC sites of the newly synthesized strand. D) HU represses the transcription of SeqA. E) DnaA binds to the DnaA boxes on the oriC region. F) when there are many DnaA molecules they repress the transcription of the dnaA gene. G) datA locus binds many DnaA molecules. 

One related control system depends on the property of DnaA to act as a transcription factor and to the presence of DnaA boxes in the promoter regions of several genes. In most cases DnaA represses the expression of the associated gene but in some cases it can activates certain genes (Messer & Weigel, 1997). DnaA regulates around 10 genes in E. coli as documented in RegulonDB (Gama-Castro et al., 2010). The transcription of DnaA is one of the most important regulatory mechanisms that directly affect the replication of DNA and one of the proteins that negatively regulate the expression of dnaA is DnaA itself (Figure 6). At high levels DnaA binds to the DnaA boxes in the promoter region and impedes transcription. This auto-repressive process directly affects the amount of DnaA-ATP available and controls the efficiency of initiation of DNA replication (Mott & Berger, 2007). In C. crescentus, it was found that DnaA also auto-represses the transcription of its own gene but additionally DnaA is highly unstable in this organism and gradually degrades after initiating a replication event (Gorbatyuk & Marczynski, 2005). 

3.2 Regulation of DNA replication by DNA methylation

 A requirement for initiation of DNA replication is that both DNA strands are methylated, principally the adenine nucleotide in the GATC motifs, this process is mediated by Dam (DNA adenine methyltranferase), (Wion & Casadésus, 2006). Dam binds to the DNA nonspecifically, and methylates the GATC motifs (Figure 6). On DNA strands recently synthesized these motifs are rapidly methylated and exist in the hemimethylated state only during a fraction of the time needed for the replication of the entire DNA (Casadésus & Low, 2006). The methylation process occurs asynchronously on the newly synthesized strands; i. e. methylation on the lagging arm occurs only after the ligation of the Okazaki fragments. It is postulated that Dam is always present in a complex bound near the replication origin, thus the methylation of nascent DNA strands occurs as soon as polymerization begins. In summary, the presence of hemimethylated GATC sites provides a cue to indicate that DNA replication has just occurred (Stancheva et al., 1999). Another way to repress the transcription of dnaA is that which occurs immediately after the initiation of DNA replication. Here, SeqA binds to the hemimethylated GATC sequences in the regulatory regions of the dnaA gene (Lu et al., 1994; Brendler et al., 2000). Similarly, SeqA also represses the replication of DNA by binding to the hemimethylated GATC sequence at the oriC, this is possible because SeqA DNA-binding sites overlap with those of low affinity for DnaA (DnaA boxes) on the oriC. This overlap impedes the complete access of DnaA-ATP to the oriC (Han et al., 2004). This prevention of replication, dependant of DNA methylation, has been considered as an epigenetic regulatory mechanism because it depends on the chemical modification of the nucleotide residues of the DNA and not in its sequence. 


Conclusions

The replication of DNA is a complex process in which a great number of regulators and mechanisms are involved, one of the most important is the DnaA protein. Replication normally begins by the formation of a complex of DnaA at the oriC region, with the assistance of DiaA, and the incorporation of some proteins that form the replisome, subsequently the formation of the open complex takes place, followed by a complex interaction of the proteins needed to execute and complete the DNA replication. The process finalizes with the recognition of the ter site and disassembly of the replisome. Many of the proteins are broadly conserved within the bacteria but some special factors are required in bacteria which undergo particular processes such as asymmetrical cell division. In general these processes are controlled by a series of circuits, which usually center on the oriC and affect the activity of DnaA. The result is regulation of the initiation step of DNA replication. Some of the regulatory mechanisms are time-dependent allowing only one DNA replication event per cell cycle. The methylation state of the DNA-strands is another important condition that not only controls the possibility of starting DNA replication but also regulates the transcription of many genes important for the execution of this function. All or certain of these mechanisms are adjusted under some special conditions, such as when the stringent response is triggered by amino acid starvation. In some bacteria with extremely reduced genomes it is still a mystery as to how DNA replication takes place and how it is controlled. Many of these latter organisms lack several important proteins implicated in the control and execution of DNA replication, and these bacteria can be useful as models for generating a system with the minimal components necessary for DNA replication.

 
1) http://cdn.intechopen.com/pdfs-wm/20627.pdf

Bacterial DNA Replication Is Coordinated with Cell Division

Bacterial cells can divide into two daughter cells at an amazing rate. Under optimal conditions, certain bacteria such as E. coli can divide every 20 to 30 minutes. DNA replication should take place only when a cell is about to divide. If DNA replication occurs too frequently, too many copies of the bacterial chromosome will be found in each cell. Alternatively, if DNA replication does not occur frequently enough, a daughter cell will be left without a chromosome. Therefore, cell division in bacterial cells must be coordinated with DNA replication. Bacterial cells regulate the DNA replication process by controlling the initiation of replication at oriC. This control has been extensively studied in E. coli. In this bacterium, several different mechanisms may control DNA replication. In general, the regulation prevents the premature initiation of DNA replication at oriC. After the initiation of DNA replication, DnaA protein hydrolyzes its ATP and therefore switches to an ADP-bound form. DnaA-ADP has a lower affinity for DnaA boxes and does not readily form a complex. This prevents premature initiation. In addition, the initiation of replication is controlled by the amount of the DnaA protein (Figure 11.17 ).


FIGURE 11.17 The amount of DnaA protein provides a way to regulate DNA replication. To begin replication, enough DnaA protein must be present to bind to all of the DnaA boxes. Immediately after DNA replication, insufficient DnaA protein is available to reinitiate a second (premature) round of DNA replication at the two origins of replication. This is because twice as many DnaA boxes are found after DNA replication and because some DnaA proteins may be degraded or stuck to other chromosomal sites and to the cell membrane.


To initiate DNA replication, the concentration of the DnaA protein must be high enough so it can bind to all of the DnaA boxes and form a complex. Immediately following DNA replication, the number of DnaA boxes is double, so an insufficient amount of DnaA protein is available to initiate a second round of replication. Also, some of the DnaA protein may be rapidly degraded and some of it may be inactive because it becomes attached to other regions of chromosomal DNA and to the cell membrane during cell division. Because it takes time to accumulate newly made DnaA protein, DNA replication cannot occur until the daughter cells have had time to grow.
Another way to regulate DNA replication involves the GATC methylation sites within oriC. These sites can be methylated by an enzyme known as DNA adenine methyltransferase (Dam). The Dam enzyme recognizes the 5ʹ–GATC–3ʹ sequence, binds there, and attaches a methyl group onto the adenine base, forming methyladenine (Figure 11.18a ).


FIGURE 11.18 Methylation of GATC sites in oriC. (a) The action of Dam (DNA adenine methyltransferase), which covalently attaches a methyl group to adenine to form methyladenine (Ame). (b) Prior to DNA replication, the action of Dam causes both adenines within the GATC sites to be methylated. After DNA replication, only the adenines in the original strands are methylated. Several minutes will pass before Dam methylates these unmethylated adenines.


DNA methylation within oriC helps regulate the replication process. Prior to DNA replication, these sites are methylated in both strands. This full methylation of the 5ʹ–GATC–3ʹ sites facilitates the initiation of DNA replication at the origin. Following DNA replication, the newly made strands are not methylated, because adenine rather than methyladenine is incorporated into the daughter strands (Figure 11.18b). The initiation of DNA replication at the origin does not readily occur until after it has become fully methylated. Because it takes several minutes for Dam to methylate the 5ʹ–GATC–3ʹ sequences within this region, DNA replication does not occur again too quickly.

http://www.nature.com/nature/journal/v421/n6921/full/nature01407.html

Mere Creation, William Dembski, starting from page 182


https://books.google.com.br/books?id=uD6KDrWLSu0C&pg=PA188&dq=DNA+Polymerase+III,+intelligent+design&hl=en&sa=X&ved=0ahUKEwiDrPucl67JAhWTqpAKHZjQCNoQ6AEIHDAA#v=onepage&q=DNA%20Polymerase%20III%2C%20intelligent%20design&f=false










  



















DNA replication is an enormously complex process with many different components that interact to ensure the faithful passing down of genetic components that interact to ensure the faithful passing down of genetic information to the next generation. A large number of parts have to work together to that end. In the absence of one or more of a number of the components, DNA replication is either halted completely or significantly compromised, and the cell either dies or becomes quite sick. Many of the components of the replication machinery form conceptually discrete subassemblies with conceptually discrete functions. The DNA polymerase holoenzyme alone would not be able to duplicate the long DNA faithfully. Tests have shown that Polymerase III alone gets stuck. Furthermore, Polymerase III is not a simple enzyme. Its rather three enzymes in one. Beside replicating DNA, it can also degrade DNA in two different ways. It does so by three different, discrete regions of the molecule. The exonuclease activity plays a critical role in replication. It allows the enzyme to proofread the new DNA and cut out any mistakes it has made. Although the polymerase reads the sequence of the old DNA to produce a new DNA, it turns out that simple base bairing allows about one mistake per thousand base pairs copied. Proofreading reduces errors to about one mistake in a million base pairs. The question is if wheter  a proofreading exonuclease and other DNA repair mechanisms had to be present in the very first cell. 

Eigen’s theory revealed the existence of the fundamental limit on the fidelity of replication (the Eigen threshold): If the product of the error (mutation) rate and the information capacity (genome size) is below the Eigen threshold, there will be stable inheritance and hence evolution; however, if it is above the threshold, the mutational meltdown and extinction become inevitable (Eigen, 1971). The Eigen threshold lies somewhere between 1 and 10 mutations per round of replication (Tejero, et al., 2011) regardless of the exact value, staying above the threshold fidelity is required for sustainable replication and so is a prerequisite for the start of biological evolution. Indeed, the very origin of the first organisms presents at least an appearance of a paradox because a certain minimum level of complexity is required to make self-replication possible at all; high-fidelity replication requires additional functionalities that need even more information to be encoded (Penny, 2005). The crucial question in the study of the origin of life is how the Darwin-Eigen cycle started—how was the minimum complexity that is required to achieve the minimally acceptable replication fidelity attained? In even the simplest modern systems, such as RNA viruses with the replication fidelity of only about 10^3 and viroids that replicate with the lowest fidelity among the known replicons (about 10^2; Gago, et al., 2009), replication is catalyzed by complex protein polymerases. The replicase itself is produced by translation of the respective mRNA(s), which is mediated by the immensely complex ribosomal apparatus. Hence, the dramatic paradox of the origin of life is that, to attain the minimum complexity required for a biological system to start on the Darwin-Eigen spiral, a system of a far greater complexity appears to be required. How such a system could evolve is a  puzzle that defeats conventional evolutionary thinking, all of which is about biological systems moving along the spiral; the solution is bound to be unusual.
That is, could the first cell, with its required complement of genes coded for by DNA, have successfully reproduced for a significant number of generations without a proofreading function ? A further question is how the function of synthesis of the lagging strand could have arisen, and the machinery to do so. That is, the Primosome, and the function of Polymerase I to remove the short peaces of RNA that the cell uses to prime replication, allowing the polymerase III function to fill the gap. These functions all require precise regulation, and coordinated functional machinelike steps. These are all complex, advanced functions and had to be present right from the beginning. How could this complex machinery have emerged in a gradual manner ? the Primosome had to be fully functional, otherwise polymerisation could not have started, since a prime sequence is required.

Clamp protein and loader. 
Most enzymes work by colliding with their substrate, catalyzing a reaction and dissociating from the product. If that were the case with DNA polymerase, then it would bind to DNA, add a nucleotide to the new chain that was being made, and then fall off of the chain. Then ,put the next nucleotide onto the growing end,  bind it and catalyze the addition. This same cycle would have to repeat itself a very large number of times to complete a new DNA chain. Modern polymerases however catalyze the addition of a nucleotide but do not fall off the DNA. Rather, they stay bound to it, until the next nucleotide comes in, and then they catalyze its addition to the chain. and they again stay bound. If it were not so, the replication process would be very slow. In the cell, polymerases stay on the DNA until their job is completed, which might be only after millions of nucleotides have been joined. This velocity is only possible because of clamp proteins. These have a ring shape. The ring can be opened up.  These clamp proteins are joined to the DNA polymerase in a intricate way, through a clamp loader protein, which has a remarkable shape similar to a human hand. It takes the clamp, like a hand with five fingers would grab it, opens it up becoming like a doughnut shape,where the whole hole in the middle is big enough to accommodate the DNA,  and then, when it is on the DNA, it positions it in a precise manner on the DNA polymerase, where it stays bound until it reaches the end of its polymerizing job. Through this ingenious process, the clamp stabilizes the DNA, making it possible to increase the speed of polymerization dramatically. Question : How would and could natural , unguided processes have figured out 1. the requirement of high-speed of polymerization ? How could they have figured out the right configuration and process to do so ? how could natural processes emerged with the right proteins incrementally, with the hand-shaped clamp loader, and the precisely fitting clamp, enabling the fast process ?? Even the most intelligent scientists are still not able to imagine how this process is engineered. Furthermore, the process requires molecular energy in the form of ATP, and everything must fit together, and be functional. Without the clamp loader protein, the clamp could not be positioned to the Polymerase enzyme, and processivity would not rise to the required speed. The whole process must also be regulated and controlled. How could that regulation have been programmed ? Trial and error ?  

DNA is a double helix. In order to read the information in DNA, the cell has to pull apart the two strands so that the new strand it is synthesizing can physically interact with the template DNA. Pulling apart the strand creates a number of problems. First of all, the two strands it is synthesizing can physically interact with the template DNA. Pulling apart the strands creates a number of problems. First of all, the two strands like to be together - they stick to each other just as if they had tiny magnets up and down their length. In order to pull apart the DNA you have to put energy into the system. In modern cells, a protein called DNA-A  binds to a specific spot along the DNA, and the protein proceeds to open up the double strand. The protein is a monomer, has motifs to bind to unique monomer sites, also they have motifs for protein-protein interaction, thus they can form clusters.   They have hydrophobic regions for helical coiling and protein–protein interactions.  Binding of the monomers to DnaA-A boxes, in ATP dependent manner (proteins have ATPase activity), leads to cooperative binding of more proteins.  This clustering of proteins on DNA makes the DNA to wrap around the proteins, which induces torsional twist and it is this left handed twist that makes DNA to melt at 13-mer region and AT rich region; perhaps the negative super helical topology in this region may further facilitate the melting of the DNA. Opening or unwinding of dsDNA into single stranded region is an important event in initiation. A second protein, called helicase, now comes along. Helicase is like a snowplow; it is a molecular machine that plows down the middle of the double helix, pushing apart the two strands. this allows the polymerase and associated proteins to travel along behind it in ease and comfort. 
 There is a problem, though, with this setup. If you push apart two DNA strands they generally do not float around separately. If they are close to one another they will rapidly snap back and form a double strand again almost as soon as the helicase passes. Even if the strands are not near each other, a single strand will usually fold up and form hydrogen bonds with itself - in other words, a tangled mess. so it is not enough to push apart the two strands of DNA; there must be a way to keep the strands apart once they have been separated. In modern cells this job is done by single-strand binding proteins, or SBB. As the helicase separates the strands of DNA, SSB coats them. 



The binding of SSB prevents the strands from rejoining until the new DNA strand has been made. There is another difficulty in being a double helix. It can be illustrated with a simple example. Take two interwined shoe laces and ask a friend to hold them together at each end. Now take a pencil, insert it between the strands near one end, and start pushing it down toward the other end. As you can see, shoestrings behind the pencil become melted, in the jargon of biochemistry. The shoestrings ahead of the pencil become more and more tangled. It becomes harder and harder to push the pencil forward. 

 Helicase and polymerase encounter the same problem with DNA. It does not matter wheter you are talking about interwined strings or interwined DNA strands. The problem of tangling is the result of the topological interconnectness of the two strands. If this problem persisted for very long in a modern cell, DNA replication would grind to a halt. However, the cell contains several enzymes, called topoisomerases, to take care of the difficulty. The way in which they do so can be illustrated with a enzyme called gyrase. Gyrase binds to DNA, pulls them apart and allows a separate portion of the DNA to pass through the cut. It then reseals the cut and lets go of the DNA. This action decreases the number of twists in DNA. In modern organisms, helicase, SSB, and gyrase all are required at the replication fork. Mutants in which any of them are missing are not viable - they die. Question : Had not all three parts , the SSB binding proteins, the topoisomerase, and the helicase and the DnaC loading proteins not have to be there all at onces, otherwise, nothing goes ? They might exercise their function but their own, but then they would not replicate DNA or have function in a bigger picture. Its evident that they had to come together to provide a functional whole. 



At the replication fork, one strand is made continuously in the 5" to the 3" direction. This is called the leading strand. The other strand, called the lagging strand, is made discontinuously, in relative small pieces called Okazaki framents. A piece is made beginning at the replication fork and proceeding for a while backward away from the fork. As the replication fork proceeds further away, the polymerase making the lagging strand hops back to the new location, again makes a new DNA chain proceeding away from the fork and continues until it bumps into the beginning of the last Okazaki fragment made. When the end of the newly made fragment bumps into the beginning of the last-made fragment, several jobs have to be done to hook the two fragments together. The first job is that the primer that begins every Okazaki fragment and is made of RNA ( not DNA ) must be removed. This is the job of Polymerase I 5" to 3" activity. the gap then has to be filled in, which Pol I  does with its polymerizing activity. Finally the two ends of the fragment have to be joined together; this is the job of an enzyme called DNA ligase.  After the completion of one Okazaki fragment , the equipment has to be released, the clamp has to let go, and a new clamp has to be loaded at the beginning of the next fragment. Clearly the formation and control of the replication fork is an enormously complex process. 



Suppose a primitive polymerase were duplicated and somehow started to replicate the second strand in the opposite direction while remaining attached to the first strand -  how could that change have been directed ? 

REplication of has to be coordinated with cell division. If the two were unconnected, then many copies of genomic DNA might be present in a cell as it started to divide, which might make the mechanical process of division very difficult or downright impossible. If cell division were more rapid than DNA replication, the genetic information might be divided and lost. at the least the cell would be wasting resources making other cell structures that, when they were dividided off from the parent cell, would carry no genetic instructions to continue. A second example of problems of control is seen in the effect in modern cells of an unbalanced nucleotide pool. If the four nucleotides that are the activated precursors to cellular DNA are not kept in a fairy even ratio with each other, many more mutations are inctroduced as the DNA is copied, launching the cell on the pathway of self-distruction. 



No one has ever published a paper in the professional science literature that explains in a detailed fashion how DNA replication de novo or any of its parts might have  been produced in a naturalistic manner, without involving intelligence. 

DNA replication is an enormously complex process with many different components that interact to ensure the faithful passing down of genetic components that interact to ensure the faithful passing down of genetic information to the next generation. A large number of parts have to work together to that end. In the absence of one or more of a number of the components, DNA replication is either halted completely or significantly compromised, and the cell either dies or becomes quite sick. Many of the components of the replication machinery form conceptually discrete sub-assemblies with conceptually discrete functions.

Wiki mentions that a key feature of the DNA replication mechanism  is that it is designed to replicate relatively large genomes rapidly and with high fidelity. Part of the cellular machinery devoted to  DNA replication and DNA-repair. The regulation of DNA replication is a vital cellular process. It is controlled by a series of mechanisms. One point of control is by modulating the accessibility of replication machinery components ( called the replisome )  to the single origin (oriC) region on the DNA. DNA replication should take place only when a cell is about to divide. If DNA replication occurs too frequently, too many copies of the bacterial chromosome will be found in each cell. Alternatively, if DNA replication does not occur frequently enough, a daughter cell will be left without a chromosome. Therefore, cell division in bacterial cells must be coordinated with DNA replication.

In prokaryotes, the DNA is circular.  Replication starts at a single origin (ori C) and is bi-directional. The region of replicating DNA associated with the single origin is called a replication bubble  and consists of two replication forks moving in opposite direction around the DNA circle. During DNA replication, the two parental strands separate and each acts as a template to direct the enzyme catalysed synthesis of a new complementary daughter strand following the normal base pairing rule. At least 10 different enzymes or proteins  participate in the initiation phase of replication. Three basic steps involved in DNA replication are Initiation, elongation and termination, subdivided in eight discrete steps.

http://reasonandscience.heavenforum.org/t1849-dna-replication-of-prokaryotes#4365

Initiation phase: 

Step 1: Initiation begins, when DNA binds around an initiator protein complex DnaA with the goal to pull the two DNA strands apart. That  creates a number of problems. First of all, the two strands like to be together - they stick to each other just as if they had tiny magnets up and down their length. In order to pull apart the DNA you have to put energy into the system. In modern cells, a protein called DnaA  binds to a specific spot along the DNA, called single origin ( oriC ) and the protein proceeds to open up the double strand. The protein is a monomer, has motifs to bind to unique monomer sites, also they have motifs for protein-protein interaction, thus they can form clusters.   They have hydrophobic regions for helical coiling and protein–protein interactions.  Binding of the monomers to DnaA-A boxes, in ATP dependent manner (proteins have ATPase activity), leads to cooperative binding of more proteins.  This clustering of proteins on DNA makes the DNA to wrap around the proteins, which induces torsional twist and it is this left handed twist that makes DNA to melt at 13-mer region and AT rich region; perhaps the negative super helical topology in this region may further facilitate the melting of the DNA. Opening or unwinding of dsDNA ( double strand DNA )  into single stranded region is an important event in initiation.  

Single-strand binding protein (SSB)
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4377

The Hexameric DnaB Helicase
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4367

DnaC, and strategies for helicase recruitment and loading in bacteria
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4371

Unwinding the DNA Double Helix Requires DNA Helicases,Topoisomerases, and Single- Stranded DNA Binding Proteins
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4374


Step 2:  During DNA replication, the two strands of the double helix must unwind at each replication fork to expose the single strands to the enzymes responsible for copying them. Three classes of proteins with distinct functions facilitate this unwinding process: DNA helicases, topoisomerases, and single-stranded DNA binding proteins ( SSB's). Helicase ( DnaB ) now comes along. The helicase exposes a region of single-stranded DNA that must be kept open for copying to proceed. Helicase is like a snowplow; it is a molecular machine that plows down the middle of the double helix, pushing apart the two strands. this allows the polymerase and associated proteins to travel along behind it in ease and comfort.  DnaB helicase alone has no affinity for ssDNA ( single stranded DNA ) bound by SSB (single- stranded binding protein). Thus, entry of the DnaB helicase complex into the unwound oriC depends on DnaC, a  additional protein factor. DnaC helps or facilitates the helicase to be loaded onto ssDNA  at the replication fork in ATP dependent manner. The DnaB-DnaC complex forms a topologically open, three-tiered toroid.  DnaC remodels DnaB to produce a cleft in the helicase ring suitable for DNA passage. DnaC’s  fold is dispensable for DnaB loading and activation. DnaB possesses autoregulatory elements that control helicase loading and unwinding. Using energy derived from ATP hydrolysis, these proteins unwind the DNA double helix in advance of the replication fork, breaking the hydrogen bonds as they go. Helicase recruitment and loading in bacteria is a remarkable process. Following video shows how that works: 

https://www.youtube.com/watch?v=YzNuLsqMqyE




There is a problem, though, with this setup. If you push apart two DNA strands they generally do not float around separately. If they are close to one another they will rapidly snap back and form a double strand again almost as soon as the helicase passes. Even if the strands are not near each other, a single strand will usually fold up and form hydrogen bonds with itself - in other words, a tangled mess. So it is not enough to push apart the two strands of DNA; there must be a way to keep the strands apart once they have been separated. In modern cells this job is done by single-strand binding proteins, or SBB's. As the helicase separates the strands of DNA, SSB's bind to the single stranded DNA and coats them.   . SSB's prevent DNA from reannealing. SSB's associate to form tetramers around which the DNA is wrapped in a manner that significantly compacts the single-stranded DNA. There is another difficulty in being a double helix. The unwinding associated with DNA replication would create an intolerable amount of supercoiling and possibly tangling in the rest of the DNA. It can be illustrated with a simple example. Take two interwined shoe laces and ask a friend to hold them together at each end. Now take a pencil, insert it between the strands near one end, and start pushing it down toward the other end. As you can see, shoestrings behind the pencil become melted, in the jargon of biochemistry. The shoestrings ahead of the pencil become more and more tangled. It becomes harder and harder to push the pencil forward.  Helicase and polymerase encounter the same problem with DNA. It does not matter wheter you are talking about interwined strings or interwined DNA strands. The problem of tangling is the result of the topological interconnectness of the two strands. If this problem persisted for very long in a  cell, DNA replication would grind to a halt. However, the cell contains several enzymes, called topoisomerases, to take care of the difficulty. The way in which they do so can be illustrated with a enzyme called gyrase. Gyrase binds to DNA, pulls them apart and allows a separate portion of the DNA to pass through the cut. It then reseals the cut and lets go of the DNA. This action decreases the number of twists in DNA. The parental DNA is unwound by DNA helicases and SSB (travels in 5’-3’ direction), the resulting positive super-coiling (torsional stress) is relieved by topoisomerse I and II (DNA gyrase) by inducing transient single stranded breaks.Topoisomerases are amazing enzymes. In this topic, a video shows how they function : 

Topoisomerase II enzymes, amazing evidence of design
http://reasonandscience.heavenforum.org/t2111-topoisomerase-ii-enzymes-amazing-evidence-of-design?highlight=topoisomerase

In modern organisms, helicase, SSB, and gyrase all are required at the replication fork. Mutants in which any of them are missing are not viable - they die.

Question : Had not all three parts , the SSB binding proteins, the topoisomerase, and the helicase and the DnaC loading proteins not have to be there all at once, otherwise, nothing goes ? They might exercise their function but their own, but then they would not replicate DNA or have function in a bigger picture. Its evident that they had to come together to provide a functional whole.  What we see here are highly coordinated , goal oriented tasks with specific  movements designed to provide a specific outcome. Auto-regulation and control   that seems required beside constant energy supply through ATP enhances the difficulty to make the whole mechanism work in the right manner. All this is awe inspiring and evidences the wise guidance and intelligence required to make all this happening in the right way.

Step 3:  The enzyme DNA primase (primase, an RNA polymerase)  attaches to the DNA and synthesizes a short RNA primer to initiate synthesis of the leading strand of the first replication fork.

Elongation phase : 

Step 4: In the elongation fase, DNA polymerase III extends the RNA primer made by primase.

DNA Polymerase
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4375

DNA polymerase possesses separate catalytic sites for polymerization and degradation of nucleic acid strands. All DNA polymerases make DNA in 5’-3’ direction . A ring-shaped sliding clamp protein encircles the DNA double helix and binds to DNA polymerase, thereby allowing the DNA polymerase to slide along the DNA while remaining firmly attached to it. Most enzymes work by colliding with their substrate, catalyzing a reaction and dissociating from the product. If that were the case with DNA polymerase, then it would bind to DNA, add a nucleotide to the new chain that was being made, and then fall off of the chain. Then ,put the next nucleotide onto the growing end,  bind it and catalyze the addition. This same cycle would have to repeat itself a very large number of times to complete a new DNA chain. Polymerases however catalyze the addition of a nucleotide but do not fall off the DNA. Rather, they stay bound to it, until the next nucleotide comes in, and then they catalyze its addition to the chain. and they again stay bound. If it were not so, the replication process would be very slow. In the cell, polymerases stay on the DNA until their job is completed, which might be only after millions of nucleotides have been joined. This velocity is only possible because of clamp proteins. These have a ring shape. The ring can be opened up.  These clamp proteins are joined to the DNA polymerase in a intricate way, through a clamp loader protein, which has a remarkable shape similar to a human hand. It takes the clamp, like a hand with five fingers would grab it, opens it up becoming like a doughnut shape,where the whole hole in the middle is big enough to accommodate the DNA,  and then, when it is on the DNA, it positions it in a precise manner on the DNA polymerase, where it stays bound until it reaches the end of its polymerizing job. Through this ingenious process, the clamp stabilizes the DNA, making it possible to increase the speed of polymerization dramatically.  They can be seen here:

The sliding clamp and clamp loader
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4376

Question : How would and could natural , unguided processes have figured out 1. the requirement of high-speed of polymerization ? How could they have figured out the right configuration and process to do so ? how could natural processes have emerged with the right proteins incrementally, with the hand-shaped clamp loader, and the precisely fitting clamp , enabling the fast process ?? Even the most intelligent scientists are still not able to imagine how this process is engineered ?  Furthermore, the process requires molecular energy in the form of ATP, and everything must fit together, and be functional. Without the clamp loader protein, the clamp could not be positioned to the polymerase enzyme, and processivity would not rise to the required speed. The whole process must also be regulated and controlled. How could that regulation have been programmed ? Trial and error ? 

Several Proteins Are Required for DNA Replication at the Replication Fork
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4398

The various proteins involved in DNA replication are all closely associated in one large complex, called a replisome.  
Leading strand synthesis:  On the template strand with 3’-5’ orientation, new DNA is made continuously in 5’-3’ direction towards the replication fork. The new strand that is continuously synthesized in 5’-3’ direction is the leading strand. 
Lagging strand synthesis: In the lagging strand, the synthesis of DNA also elongates in a 5ʹ to 3ʹ manner, but it does so in the direction away from the replication fork. In the lagging strand, RNA primers must repeatedly initiate the synthesis of short segments of DNA; thus, the synthesis has to be discontinuous.

The Primase (DnaG) enzyme, and the primosome complex
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4379

 The length of these fragments in bacteria is typically 1000 to 2000 nucleotides. In eukaryotes, the fragments are shorter—100 to 200 nucleotides. Each fragment contains a short RNA primer at the 5ʹ end, which is made by primase. The remainder of the fragment is a strand of DNA made by DNA polymerase III. The DNA fragments made in this manner are known as Okazaki fragments. To complete the synthesis of Okazaki fragments within the lagging strand, three additional events must occur: removal of the RNA primers, synthesis of DNA in the area where the primers have been removed, and the covalent attachment of adjacent fragments of DNA. In E. coli, the RNA primers are removed by the action of DNA polymerase I. This enzyme has a 5ʹ to 3ʹ exonuclease activity, which means that DNA polymerase I digests away the RNA primers in a 5ʹ to 3ʹ direction, leaving a vacant area. DNA polymerase I then synthesizes DNA to fill in this region. It uses the 3ʹ end of an adjacent Okazaki fragment as a primer. , DNA polymerase I would remove the RNA primer from the first Okazaki fragment and then synthesize DNA in the vacant region by attaching nucleotides to the 3ʹ end of the second Okazaki fragment. After the gap has been completely filled in, a covalent bond is still missing between the last nucleotide added by DNA polymerase I and the adjacent DNA strand that had been previously made by DNA polymerase III. To the left of the origin, the top strand is made continuously, whereas to the right of the origin it is made in Okazaki fragments. By comparison, the synthesis of the bottom strand is just the opposite. To the left of the origin it is made in Okazaki fragments and to the right of the origin the synthesis is continuous. Finally the two ends of the fragment have to be joined together; this is the job of an enzyme called DNA ligase.  After the completion of one Okazaki fragment , the equipment has to be released, the clamp has to let go, and a new clamp has to be loaded at the beginning of the next fragment. Clearly the formation and control of the replication fork is an enormously complex process. 

Step 5:   After DNA synthesis by DNA pol III, DNA polymerase I uses its 5’-3’ exonuclease activity to remove the RNA primer and fills the gaps with new DNA. In the next step, finally DNA ligase joins the ends of the DNA fragments together.  As the replisome moves along the DNA in the direction of the replication fork, it must accommodate the fact that DNA is being synthesized in opposite directions along the template on the two stands. Picture above  provides a schematic model illustrating how this might be accomplished by folding the lagging strand template into a loop.Creating such a loop allows the DNA polymerase molecules on both the leading and lagging strands to move in the same physical direction, even though the two template strands are oriented with opposite polarity. The replisome faces special challenges as it makes new DNA at rates that can approach 1,000 nucleotides per second. Unlike the machines that make proteins and RNA, which work relatively sluggishly and in a linear fashion, the replisome must simultaneously copy two strands of DNA that are aligned in opposite directions (5ʹ to 3ʹ and 3ʹ to 5ʹ). Replisome chemistry obeys two rules. 


Questions: How did they arise with that cabability to " obey two rules " ?  Suppose a primitive polymerase were duplicated and somehow started to replicate the second strand in the opposite direction while remaining attached to the first strand -  how could that change have been directed , and why should that feat have happened randomly ? 




The DNA polymerase holoenzyme alone would not be able to duplicate the long DNA faithfully. Tests have shown that Polymerase III alone gets stuck. Furthermore, Polymerase III is not a simple enzyme. Its rather three enzymes in one. Beside replicating DNA, it can also degrade DNA in two different ways. It does so by three different, discrete regions of the molecule. The exonuclease activity plays a critical role in replication. It allows the enzyme to proofread the new DNA and cut out any mistakes it has made. Although the polymerase reads the sequence of the old DNA to produce a new DNA, it turns out that simple base bairing allows about one mistake per thousand base pairs copied. Proofreading reduces errors to about one mistake in a million base pairs. The question is if wheter  a proofreading exonuclease and other DNA repair mechanisms had to be present in the very first cell. 

Eigen’s theory revealed the existence of the fundamental limit on the fidelity of replication (the Eigen threshold): If the product of the error (mutation) rate and the information capacity (genome size) is below the Eigen threshold, there will be stable inheritance and hence evolution; however, if it is above the threshold, the mutational meltdown and extinction become inevitable (Eigen, 1971). The Eigen threshold lies somewhere between 1 and 10 mutations per round of replication (Tejero, et al., 2011) regardless of the exact value, staying above the threshold fidelity is required for sustainable replication and so is a prerequisite for the start of biological evolution. Indeed, the very origin of the first organisms presents at least an appearance of a paradox because a certain minimum level of complexity is required to make self-replication possible at all; high-fidelity replication requires additional functionalities that need even more information to be encoded (Penny, 2005). The crucial question in the study of the origin of life is how the Darwin-Eigen cycle started—how was the minimum complexity that is required to achieve the minimally acceptable replication fidelity attained? In even the simplest modern systems, such as RNA viruses with the replication fidelity of only about 10^3 and viroids that replicate with the lowest fidelity among the known replicons (about 10^2; Gago, et al., 2009), replication is catalyzed by complex protein polymerases. The replicase itself is produced by translation of the respective mRNA(s), which is mediated by the immensely complex ribosomal apparatus. Hence, the dramatic paradox of the origin of life is that, to attain the minimum complexity required for a biological system to start on the Darwin-Eigen spiral, a system of a far greater complexity appears to be required. How such a system could evolve is a  puzzle that defeats conventional evolutionary thinking, all of which is about biological systems moving along the spiral; the solution is bound to be unusual. 

DNA damage and repair
http://reasonandscience.heavenforum.org/t2043-dna-repair?highlight=dna+repair
http://reasonandscience.heavenforum.org/t1849p30-dna-replication-of-prokaryotes#4401


Replication forks may stall frequently and require some form of repair to allow completion of chromosomal duplication. Failure to solve these replicative problems comes at a high price, with the consequences being genome instability, cell death and, in higher organisms, cancer. Replication fork repair and hence reloading of DnaB may be needed away from oriC at any point within the chromosome and at any stage during chromosomal duplication. The potentially catastrophic effects of uncontrolled initiation of chromosomal duplication on genome stability suggests that replication restart must be regulated as tightly as DnaA-directed replication initiation at oriC. This implies reloading of DnaB must occur only on ssDNA at repaired forks or D-loops rather than onto other regions of ssDNA, such as those created by blocks to lagging strand synthesis.Thus an alternative replication initiator protein, PriA helicase, is utilized during replication restart to reload DnaB back onto the chromosome 

Question: Could the first cell, with its required complement of genes coded for by DNA, have successfully reproduced for a significant number of generations without a proofreading function ? A further question is how the function of synthesis of the lagging strand could have arisen, and the machinery to do so. That is, the Primosome, and the function of Polymerase I to remove the short peaces of RNA that the cell uses to prime replication, allowing the polymerase III function to fill the gap. These functions all require precise regulation, and coordinated functional machine-like steps. These are all complex, advanced functions and had to be present right from the beginning. How could this complex machinery have emerged in a gradual manner ? the Primosome had to be fully functional, otherwise polymerisation could not have started, since a prime sequence is required.

Step 6: Finally DNA ligase joins the ends of the DNA fragments together.

Termination phase: 

Termination of DNA replication
http://reasonandscience.heavenforum.org/t1849p15-dna-replication-of-prokaryotes#4399

Step 7: The two replication forks meet ~ 180  degree opposite to ori C, as DNA is circular in prokaryotes. Around this region there are several terminator sites which arrest the movement of forks by binding to the tus gene product, an inhibitor of helicase (Dna B).
Step 8: Once replication is complete, the two double stranded circular DNA molecules (daughter strands) remain interlinked. Topoisomerase II makes double stranded cuts to unlink these molecules.

I do not know of any scientific paper  that explains in a detailed manner how DNA replication de novo or any of its parts might have emerged in a naturalistic manner, without involving intelligence. The systems responsible for DNA replication are well beyond the explanatory power of unguided natural processes without guiding intelligence involved. Indeed, machinery of the complexity and sophistication of that described above is, is in my view best explained through a intelligent designer.

Recombination Vital to Genome Stability  

The latest issue of the Proceedings of the National Academy of Sciences (July 17) contains a symposium on gene replication and recombination, among other papers on DNA.  Among the interesting papers:
(1) A theory on how genomes can contain vast stretches of non-coding DNA, apparently inactive retrotransposons that were inserted by recombination, polyploidy or lateral transfer.  These inactive stretches, while harmless, can greatly expand the genome while keeping the number of actual genes relatively constant.  
(2) A description of how recombination is an essential method for repair of DNA breaks, stating that “DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins.  Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human.”  
(3) Another paper describes how specialized proteins called topoisomerases help prevent the strain of uncoiling DNA from breaking,  but when they fail, recombination can help restart the replication process.  
(4) A paper describes how recombination works to repair breaks in a replicating chromosome.  
(5) Some Japanese scientists describe how a gene codes for a motor protein that is essential for genome stability.
(6) The cover story describes the various repair mechanisms, stating, “Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes.  Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases.” Describing one such mechanism named Rad52, the authors state, “The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands . . . . our data indicate that each Rad52 focus [i.e. active site] represents a center of recombinational repair capable of processing multiple DNA lesions.”


These are just samples of the exciting findings being made about DNA replication.  These and other papers show that it is a fail-safe system with many sophisticated backup and repair mechanisms.  While there is still much to learn, and many mysteries to explain, DNA’s ability to replicate is truly a marvel of engineering.  Think about the classic chicken-and-egg conundrum for evolution illustrated by (5) above: a gene codes for a protein that is essential for the gene to exist.  Browse through the abstracts of these papers just to get a feel for the amazingly complex world of cellular processes going on in your body right now, without your conscious thought or control.


http://creationsafaris.com/crev07.htm

Eugene Koonin, in the following peer-reviewed article : The cosmological model of eternal inflation and the transition from chance to biological evolution in the history of life

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1892545/

wrote following:

“However, it is clear that origin(s) of replication and translation (OORT) is not just the hardest problem in all of evolutionary biology but one that is qualitatively distinct from the rest. For all other problems, the basis of biological evolution, genome replication, is in place but, in the case of OORT, the emergence of this mechanism itself is the explanandum. Thus, it is of interest to consider radically different scenarios for OORT.”

He calculates that, using a “toy model that assumes a deliberately inflated rate of RNA production, the probability that a coupled translation-replication emerges by chance...is < 10-10^18.”

He recognises that such a low probability (which of course can be questioned) requires an explanation beyond chance in our universe, and in order to avoid the design hypothesis, proposes a pseudo scientific multiverse.

“Despite considerable experimental and theoretical effort, no compelling scenarios currently exist for the origin of replication and translation, the key processes that together comprise the core of biological systems and the apparent pre-requisite of biological evolution. The RNA World concept might offer the best chance for the resolution of this conundrum but so far cannot adequately account for the emergence of an efficient RNA replicase or the translation system."

“The MWO version of the cosmological model of eternal inflation could suggest a way out of this conundrum because, in an infinite multiverse with a finite number of distinct macroscopic histories (each repeated an infinite number of times), emergence of even highly complex systems by chance is not just possible but inevitable.”

Thats a nice example of how metaphysical just so speculation is accepted as peer reviewed science. Crazy.

When the truth is rejected, any kind of nonsense replaces it.

DNA replication, and its nanotechnology that defies naturalistic explanations

The Argument of the Original Replicator
In prokaryotic cells, DNA replication involves more than thirty specialized proteins to perform tasks necessary for building and accurately copying the genetic molecule.
Each of these proteins is essential and required for the proper replicating process. Not a single one of these proteins can be missing, otherwise, the whole process breaks
down, and is unable to perform its task correctly. DNA repair mechanisms must also be in place,  fully functional and working properly, otherwise, the mutation
rate will be too high, and the cell dies. 
The individual parts and proteins require by themselves complex assembly proteins to be built.
The individual parts, assembly proteins, and proteins individually would have no function on their own. They have only function interconnected in the working whole.
The individual parts must be readily available on the construction site of the RNA replication complex, being correctly interlocked, interlinked, and have the right interface
compatibility to be able to interact correctly together. All this requires information and meta information ( information that directs the expression of the
genomic information for construction of the individual proteins, and correct timing of expression, and as well the information of the correct assembly sequence. )
Evolution is not a capable driving force to make the DNA replicating complex, because evolution depends on cell replication through the very own mechanism we try
to explain. It takes proteins to make DNA replication happen. But it takes the DNA replication process to make proteins. That’s a catch 22 situation.
DNA replication requires coded, complex, specified information and meta-information, and the DNA replication process is irreducibly complex.
Therefore, DNA replication is best explained through design.

The Argument of the Original Replicator

1. In prokaryotic cells, DNA replication involves more than thirty specialized proteins to perform tasks necessary for building and accurately copying the genetic molecule.
2. Each of these proteins is required for the proper replicating process. Not a single one of them can be missing, otherwise the whole process breaks down.
3. DNA repair mechanisms must also be in place, fully functional and working properly, otherwise the mutation rate will be too high, and the cell dies.
4. The individual parts and proteins require by themselves complex assembly proteins to be built.
5. The individual parts, assembly proteins, and proteins individually would have no function by their own. They have only function interconnected in the working whole.
6. The individual parts must be readily available on the construction site of the DNA replication complex, being correctly interlocked, interlinked, and have the right interface compatibility to be able to interact correctly together. All this requires information and meta information ( information that directs the expression of the genomic information for construction of the individual proteins, and correct timing of expression, and as well the information of the correct assembly sequence. )
7. Evolution is not a capable driving force to make the dna replicating complex, because evolution depends on cell replication through the very own mechanism we try to explain.
8. It takes proteins to make DNA replication happen. But it takes the DNA replication process to make proteins. That’s a catch 22 situation.
9. DNA replication requires coded, complex, specified information and meta-information, and the DNA replication process is irreducibly complex.
10 Therefore, DNA replication is best explained through set up by an intelligent designer.

There are currently 593 proteins known to be required to assure high fidelity of human DNA replication and to prevent disease.
What a stunning revelation! Without these proteins operating in concert, our DNA would degenerate rapidly and completely within a matter of a few generations. The vital question...how did these proteins, required for DNA replication emerge in the first place. They are paramount to intelligent design. Performing seeming miracles with every cell division.


They found that 85% of the proteins had activities consistent with a function in DNA and chromatin replication or replication stress responses.

https://phys.org/news/2019-10-dna-replication-proteins.html?fbclid=IwAR15mW8XVT1UfLyJpACsrvGDW9ETtdjHAqFHDpURY5tJjLP3-PgpyCQds90

Determination of the Core of a Minimal Bacterial Gene Set Sept. 2004
https://mmbr.asm.org/content/mmbr/68/3/518.full.pdf


DNA metabolism. (i) Basic replication machinery.
One of the most important housekeeping functions in all living cells is DNA replication, a multistage reaction with four basic steps. 

First, a replication origin sequence within the genome is recognized by protein components. 
Second, initiation of replication occurs through the recruitment of replisomal proteins at the origin. 
Third, the general replication reaction duplicates both strands of DNA. 
Fourth, replication terminates and the two daughter chromosomes are separated. 

The specific mechanism for replication initiation depends both on the structure of the replication origin and on the nature of the initiation protein. Several mechanisms have been described in bacteria, but the analysis of the genome of B. floridanus, the endosymbiotic bacteria of carpenter ants, revealed that it does not encode any of the proposed initiation proteins (DnaA, RecA, and PriA). Another recruitment protein (DnaC in E. coli, DnaI in B. subtilis) is also required to form a complex with the helicase DnaB (EC 3.6.1.-) to prevent the indiscriminate binding of the helicase to single-stranded DNA, transferring it only in the correct DNA location which is not coated by Single-Stranded DNA Binding protein. (SSB). However, DnaC is absent in B. floridanus and W. glossinidia (endosymbiont of tsetse flies), indicating that it is also dispensable in these bacteria. Hence, it can be assumed that DNA replication can take place without the need for these initiation and recruiting proteins under some conditions, which might be the case for small genomes, or that there are other, as yet unidentified proteins that perform such functions in these reduced genomes. The recruitment and loading of helicase at the replication origin generally requires the presence of a histonelike protein for the destabilization of the duplex DNA near the origin of replication. At least one histone-like protein is also encoded by all five analyzed endosymbiont genomes, although none of these proteins is conserved in all of them. In M. genitalium, MG353 appears to encode a nucleoid DNA binding protein similar to hupA. Since this gene is also present in all endosymbionts analyzed except B. floridanus, we decided to include this histone-like protein-coding gene in our minimal set. To begin DNA replication, the helicase DnaB (EC 3.6.1.-) attracts the primase DnaG (EC 2.7.7.-) to the replication fork, both of them present in all bacteria with reduced genomes analyzed in this study. Once the DNA is melted and primed, the general DNA replication begins, with the action of DNA polymerase III (EC 2.7.7.7). Genes encoding the  subunits of DNA polymerase III, all subunits that are present both in gram-negative and gram-positive bacteria, are also conserved. It should be noticed that in gram-positive bacteria there are two genes encoding the subunits of this holoenzyme, both of them essential in B. subtilis, but one of them (dnaE) has been proven to be dispensable in M. genitalium, while polC encodes both the and ε subunits of DNA polymerase III. Two more proteins required at this stage, gyrase (EC 5.99.1.2, encoded by gyrA and gyrB) and ligase (EC 6.5.1.2, encoded by lig), are also conserved, thus allowing the completion of the third stage of DNA replication. Although lig was previously annotated as a pseudogene in B. aphidicola BSg due to the absence of the C-terminal protein end (80), it has been reannotated as a functional gene encoding the catalytic domain of the protein but with the absence of the BRCT domain (28). On the other hand, the gene encoding the protein necessary to stop replication by inhibiting helicase translocation (tus in E. coli, rtp in B. subtilis) cannot be found in the reduced genomes under study. Several topoisomerases that are present in M. genitalium and essential in B. subtilis and E. coli to allow independent segregation of the two daughter chromosomes (EC 5.99.1.2, encoded by topA, and EC 5.99.1.-, encoded by parC and parE) must also be considered dispensable, since parC and parE have been lost in all endosymbionts analyzed, while topA is only present in two strains of B. aphidicola. It is possible that the above-mentioned GyrA/B, which is present in all these bacteria, performs all the topoisomerase functions required for DNA replication and segregation, since it has been suggested that type IA (TopA) and type II (GyrA/B and ParD/E) topoisomerases may use comparable mechanisms to bind and cleave DNA, based on similarities in several structural elements (6). Other proteins involved in chromosome condensation that are essential in B. subtilis (SMC, ScpA, and ScpB) are not conserved either, and they have not been included in the minimal set. SMC proteins are conserved in most (but not all) prokaryotes, and E. coli mutants lacking the encoding gene are viable.