The Cell cycle

Part one of this series looked at the cycles within cycles that make up the existence of a cell. Whilst taking up such a small percentage of the overall cell cycle, mitosis is one of the most important series of events in the life of a cell.  1

In addition to duplicating their genome, most cells also duplicate their other organelles and macromolecules; otherwise, daughter cells would get smaller with each division. To maintain their size, dividing cells must coordinate their growth (that is, their increase in cell mass) with their division.  We begin with a brief overview of the cell cycle. We then describe the cell-cycle control system, a complex network of regulatory proteins that triggers the different events of the cycle. We next consider in detail the major stages of the cell cycle, in which the chromosomes are duplicated and then segregated into the two daughter cells. Finally, we consider how extracellular signals govern the rates of cell growth and division and how these two processes are coordinated.

For many years, cell biologists watched the puppet show of DNA synthesis, mitosis, and cytokinesis but had no idea of what lay behind the curtain controlling these events. It was not even clear whether there was a separate control system, or whether the processes of DNA synthesis, mitosis, and cytokinesis somehow controlled themselves. A major breakthrough came in the late 1980s with the identification of the key proteins of the control system, along with the realization that they are distinct from the proteins that perform the processes of DNA replication, chromosome segregation, and so on. In this section, we first consider the basic principles upon which the cell-cycle control system operates. We then discuss the protein components of the system and how they work together to time and coordinate the events of the cell cycle.

The Cell-Cycle Control System Triggers the Major Events of the Cell Cycle



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The cell-cycle control system operates much like a timer that triggers the events of the cell cycle in a set sequence



In its simplest form the control system is rigidly programmed to provide a fixed amount of time for the completion of each cell-cycle event.  The control system in these early embryonic divisions is independent of the events it controls, so that its timing mechanisms continue to operate even if those events fail. In most cells, however, the control system does respond to information received back from the processes it controls If some malfunction prevents the successful completion of DNA synthesis, for example, signals are sent to the control system to delay progression to M phase. Such delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage—and segregated incompletely replicated chromosomes, for example. The cell-cycle control system is based on a connected series of biochemical switches, each of which initiates a specific cell-cycle event. This system of switches possesses many important features that increase the accuracy and reliability of cell-cycle progression. First, the switches are generally binary (on/off) and launch events in a complete, irreversible fashion. It would clearly be disastrous, for example, if events like chromosome condensation or nuclear-envelope breakdown were only partially initiated or started but not completed. Second, the cell-cycle control system is remarkably robust and reliable, partly because backup mechanisms and other features allow the system to operate effectively under a variety of conditions and even if some components fail. Finally, the control system is highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellular signals.

The Cell-Cycle Control System Depends on Cyclically Activated Cyclin-Dependent Protein Kinases (Cdks)

Central components of the cell-cycle control system are members of a family of protein kinases known as  Cyclin-dependent kinases (Cdks). The activities of these kinases rise and fall as the cell progresses through the cycle, leading to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle. An increase in Cdk activity at the G2/M transition, for example, increases the Phosphorylation of proteins that control chromosome condensation, nuclear-envelope breakdown, spindle assembly, and other events that occur in early mitosis. Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins. The most important of these Cdk regulators are proteins known as cyclins. Cdks, as their name implies, are dependent on cyclins for their activity: unless they are bound tightly to a cyclin, they have no protein kinase activity.



CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. 1



Cyclins were originally named because they undergo a cycle of synthesis and degradation in each cell cycle. The levels of the Cdk proteins, by contrast, are constant. Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin–Cdk complexes at specific stages of the cell cycle.

Cyclins 2 are generally very different from each other in primary structure, or amino acid sequence. However, all members of the cyclin family are similar in 100 amino acids that make up the cyclin box. Cyclins contain two domains of similar all-α fold, the first located at the N-terminus and the second at the C-terminus. All cyclins are believed to contain a similar tertiary structure of two compact domains of 5 α helices. The first of which is the conserved cyclin box, outside of which cyclins are divergent. For example, the amino-terminal regions of S and M cyclins contain short destruction-box motifs that target these proteins for proteolysis in mitosis.



Cyclins were originally named because they undergo a cycle of synthesis and degradation in each cell cycle. The levels of the Cdk proteins, by contrast, are constant. Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin–Cdk complexes at specific stages of the cell cycle. There are four classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function. All eukaryotic cells require three of these classes



1. G1/S-cyclins activate Cdks in late G1 and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase.

2. S-cyclins bind Cdks soon after progression through Start and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events.

3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid-mitosis. In most cells, a fourth class of cyclins, the G1-cyclins, helps govern the activities of the G1/S-cyclins, which control progression through Start in late G1. In yeast cells, a single Cdk protein binds all classes of cyclins and triggers different cell-cycle events by changing cyclin partners at different stages of the cycle. In vertebrate cells, by contrast, there are four Cdks. Two interact with G1-cyclins, one with G1/S- and S-cyclins, and one with S- and M-cyclins. In this chapter, we simply refer to the different cyclin–Cdk complexes as G1-Cdk, G1/S-Cdk, S-Cdk, and M-Cdk.

How do different cyclin–Cdk complexes trigger different cell-cycle events? The answer, at least in part, seems to be that the cyclin protein does not simply activate its Cdk partner but also directs it to specific target proteins. As a result, each cyclin–Cdk complex phosphorylates a different set of substrate proteins. The same cyclin–Cdk complex can also induce different effects at different times in the cycle, probably because the accessibility of some Cdk substrates changes during the cell cycle. Certain proteins that function in mitosis, for example, may become available for phosphorylation only in G2.

Studies of the three-dimensional structures of Cdk and cyclin proteins have revealed that, in the absence of cyclin, the active site in the Cdk protein is partly obscured by a protein loop, like a stone blocking the entrance to a cave (Figure A). Cyclin binding causes the loop to move away from the active site, resulting in partial activation of the Cdk enzyme (Figure B). Full activation of the cyclin–Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site. This causes a small conformational change that further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events (Figure C).



Cdk Activity Can Be Suppressed By Inhibitory Phosphorylation and Cdk Inhibitor Proteins (CKIs)

The rise and fall of cyclin levels is the primary determinant of Cdk activity during the cell cycle. Several additional mechanisms, however, help control Cdk activity
at specific stages of the cycle. Phosphorylation at a pair of amino acids in the roof of the kinase active site inhibits the activity of a cyclin–Cdk complex. Phosphorylation of these sites by a protein kinase known as Wee1 inhibits Cdk activity, while dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity



We will see later that this regulatory mechanism is particularly important in the control of M-Cdk activity at the onset of mitosis. Binding of Cdk inhibitor proteins (CKIs) inactivates cyclin–Cdk complexes. The three-dimensional structure of a cyclin–Cdk–CKI complex reveals that CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactive



Cells use CKIs primarily to help govern the activities of G1/S- and S-Cdks early in the cell cycle.

Regulated  proteolysis Triggers the  Metaphase to Anaphase transition


Whereas activation of specific cyclin–Cdk complexes drives progression through the Start and G2/M transitions, progression through the metaphase-to-anaphase transition is triggered not by protein phosphorylation but by protein destruction, leading to the final stages of cell division. The key regulator of the metaphase-to-anaphase transition is the anaphasepromoting complex, or cyclosome (APC/C) , a member of the ubiquitin ligase family of enzymes.



These enzymes are used in numerous cell processes to stimulate the proteolytic destruction of specific regulatory proteins. They  polyubiquitylate specific target proteins, resulting in their destruction in proteasomes. Other ubiquitin ligases mark proteins for purposes other than destruction. The APC/C catalyzes the ubiquitylation and destruction of two major types of proteins. The first is securin, which protects the protein linkages that hold sister-chromatid pairs together in early mitosis. Destruction of securin in metaphase activates a protease that separates the sisters and unleashes anaphase, as described later. The S- and M-cyclins are the second major targets of the APC/C. Destroying these cyclins inactivates most Cdks in the cell. As a result, the many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatases in the anaphase cell. This dephosphorylation of Cdk targets is required for the completion of M phase, including the final steps in mitosis and then cytokinesis. Following its activation in mid-mitosis, the APC/C remains active in G1 to provide a stable period of Cdk inactivity. When G1/S-Cdk is activated in late G1, the APC/C is turned off, thereby allowing cyclin accumulation in the next cell cycle.

The cell-cycle control system also uses another ubiquitin ligase called SCF.



It has many functions in the cell, but its major role in the cell cycle is to ubiquitylate certain CKI proteins in late G1, thereby helping to control the activation of S-Cdks and DNA replication. SCF is also responsible for the destruction of G1/S-cyclins in early S phase. The APC/C and SCF are both large, multisubunit complexes with some related components
, but they are regulated differently. APC/C activity changes during the cell cycle, primarily as a result of changes in its association with an activating subunit—either Cdc20 in mid-mitosis or Cdh1 from late mitosis through early G1. These subunits help the APC/C recognize its target proteins



(Figure A above). SCF activity depends on substrate-binding subunits called F-box proteins. Unlike APC/C activity, however, SCF activity is constant during the cell
cycle. Ubiquitylation by SCF is controlled instead by changes in the phosphorylation state of its target proteins, as F-box subunits recognize only specifically
phosphorylated proteins (Figure B).

Cell-Cycle Control Also Depends on Transcriptional Regulation

In the simple cell cycles of early animal embryos, gene transcription does not occur. Cell-cycle control depends exclusively on post-transcriptional mechanisms that involve the regulation of Cdks and ubiquitin ligases and their target proteins. In the more complex cell cycles of most cell types, however, transcriptional control provides an important additional level of regulation. Changes in cyclin gene transcription, for example, help control cyclin levels in most cells. A variety of methods  have been used to analyze changes in the expression of all genes in the genome as the cell progresses through the cell cycle. The results of these studies are surprising. In budding yeast, for example, about 10% of the genes encode mRNAs whose levels oscillate during the cell cycle. Some of these genes encode proteins with known cell-cycle functions, but the functions of many others are unknown.

Duplication of the chromosomes in S phase involves the accurate replication of the entire DNA molecule in each chromosome, as well as the duplication of the chromatin proteins that associate with the DNA and govern various aspects of chromosome function. Chromosome duplication is triggered by the activation of S-Cdk, which activates proteins that unwind the DNA and initiate its replication at replication origins. Once a replication origin is activated, S-Cdk also inhibits proteins that are required to allow that origin to initiate DNA replication again. Thus, each origin is fired once and only once in each S phase and cannot be reused until the next cell cycle.


The linear chromosomes of eukaryotic cells are vast and dynamic assemblies of DNA and protein, and their duplication is a complex process that takes up a major fraction of the cell cycle. Not only must the long DNA molecule of each chromosome be duplicated accurately—a remarkable feat in itself—but the protein packaging surrounding each region of that DNA must also be reproduced, ensuring that the daughter cells inherit all features of chromosome structure. The central event of chromosome duplication—DNA replication—poses two problems for the cell. First, replication must occur with extreme accuracy to minimize the risk of mutations in the next cell generation. Second, every nucleotide in the genome must be copied once, and only once, to prevent the damaging effects of gene amplification.  Sophisticated protein machinery  performs DNA replication with astonishing speed and accuracy. In this section, we consider the elegant mechanisms by which the cell-cycle control system initiates the replication process and, at the same time, prevents it from happening more than once per cycle.[/b]

S-Cdk Initiates DNA Replication Once Per Cycle

DNA replication begins at origins of replication, which are scattered at numerous locations in every chromosome. During S phase, DNA replication is initiated at these origins when a DNA helicase unwinds the double helix and DNA replication enzymes are loaded onto the two single-stranded templates. This leads to the elongation phase of replication, when the replication machinery moves outward from the origin at two replication forks.

To ensure that chromosome duplication occurs only once per cell cycle, the initiation phase of DNA replication is divided into two distinct steps that occur at
different times in the cell cycle



The first step occurs in late mitosis and early G1, when a pair of inactive DNA helicases is loaded onto the replication origin, forming a large complex called the prereplicative complex or preRC. This step is sometimes called licensing of replication origins because initiation of DNA synthesis is permitted only at origins containing a preRC. The second step occurs in S phase, when the DNA helicases are activated, resulting in DNA unwinding and the initiation of DNA synthesis. Once a replication origin has been fired in this way, the two helicases move out from the origin with the replication forks, and that origin cannot be reused until a new preRC is assembled there at the end of mitosis. As a result, origins can be activated only once per cell cycle.



Above figure illustrates some of the molecular details underlying the control of the two steps in the initiation of DNA replication. A key player is a large multiprotein complex called the origin recognition complex (ORC), which binds to replication origins throughout the cell cycle. In late mitosis and early G1, the proteins Cdc6 and Cdt1 collaborate with the ORC to load the inactive DNA helicases around the DNA next to the origin. The resulting large complex is the preRC, and the origin is now licensed for replication. At the onset of S phase, S-Cdk triggers origin activation by phosphorylating specific initiator proteins, which then nucleate the assembly of a large protein complex that activates the DNA helicase and recruits the DNA synthesis machinery. Another protein kinase called DDK is also activated in S phase and helps drive origin activation by phosphorylating specific subunits of the DNA helicase. At the same time as S-Cdk initiates DNA replication, several mechanisms prevent assembly of new preRCs. S-Cdk phosphorylates and thereby inhibits the ORC and Cdc6 proteins. Inactivation of the APC/C in late G1 also helps turn off preRC assembly. In late mitosis and early G1, the APC/C triggers the destruction of a Cdt1 inhibitor called geminin, thereby allowing Cdt1 to be active. When the APC/C is turned off in late G1, geminin accumulates and inhibits the Cdt1 that is not associated with DNA. Also, the association of Cdt1 with a protein at active replication forks stimulates Cdt1 destruction. In these various ways, preRC formation is prevented from S phase to mitosis, thereby ensuring that each origin is fired only once per cell cycle. How, then, is the cell-cycle control system reset to allow replication in the next cell cycle? At the end of mitosis, APC/C activation leads to the inactivation of Cdks and the destruction of geminin. ORC and Cdc6 are dephosphorylated and Cdt1 is activated, allowing preRC assembly to prepare the cell for the next S phase.

Chromosome Duplication Requires Duplication of Chromatin Structure

The DNA of the chromosomes is extensively packaged in a variety of protein components, including histones and various regulatory proteins involved in the control of gene expression. Thus, duplication of a chromosome is not simply a matter of replicating the DNA at its core but also requires the duplication of these chromatin proteins and their proper assembly on the DNA. The production of chromatin proteins increases during S phase to provide the raw materials needed to package the newly synthesized DNA. Most importantly, S-Cdks stimulate a large increase in the synthesis of the four histone subunits that form the histone octamers at the core of each nucleosome. These subunits are
assembled into nucleosomes on the DNA by nucleosome assembly factors, which typically associate with the replication fork and distribute nucleosomes on both strands of the DNA as they emerge from the DNA synthesis machinery. Chromatin packaging helps to control gene expression. In some parts of the chromosome, the chromatin is highly condensed and is called heterochromatin, whereas in other regions it has a more open structure and is called euchromatin. These differences in chromatin structure depend on a variety of mechanisms, including modification of histone tails and the presence of non-histone proteins. Because these differences are important in gene regulation,it is crucial that chromatin structure, like the DNA within, is reproduced accurately during S phase. How chromatin structure is reproduced is not well understood, however. During DNA synthesis, histone-modifying enzymes and various non-histone proteins are probably deposited onto the two new DNA strands as they emerge from the replication fork, and these proteins are thought to reproduce the local chromatin structure of the parent chromosome. 

Cohesins Hold Sister Chromatids Together

At the end of S phase, each replicated chromosome consists of a pair of identical sister chromatids glued together along their length. This sister-chromatid cohesion sets the stage for a successful mitosis because it greatly facilitates the attachment of the two sister chromatids to opposite poles of the mitotic spindle. Imagine how difficult it would be to achieve this bipolar attachment if sister chromatids were allowed to drift apart after S phase. Indeed, defects in sister-chromatid cohesion— in yeast mutants, for example—lead inevitably to major errors in chromosome segregation. Sister-chromatid cohesion depends on a large protein complex called cohesin, which is deposited at many locations along the length of each sister chromatid as the DNA is replicated in S phase. Two of the subunits of cohesin are members of a large family of proteins called SMC proteins (for Structural Maintenance of Chromosomes). Cohesin forms giant ringlike structures, and it has been proposed that these surround the two sister chromatids



Sister-chromatid cohesion also results, at least in part, from DNA catenation, the intertwining of sister DNA molecules that occurs when two replication forks meet during DNA synthesis.

The enzyme topoisomerase II gradually disentangles the catenated sister DNAs between
S phase and early mitosis by cutting one DNA molecule, passing the other through the break,
and then resealing the cut DNA



Picture below: The DNA-helix-passing reaction catalyzed by DNA topoisomerase II. Unlike type I topoisomerases, type II enzymes hydrolyze ATP (red), which is needed to release and reset the enzyme after each cycle. Type II topoisomerases are largely confined to proliferating cells in eukaryotes; partly for that reason, they have been effective targets for anticancer drugs. Some of these drugs inhibit topoisomerase II at the third step in the figure and thereby produce high levels of double-strand breaks that kill rapidly
dividing cells. The small yellow circles represent the phosphates in the DNA backbone that become covalently bonded to the topoisomerase



Topoisomerase II forms a covalent linkage to both strands of the DNA helix at the same time, making a transient double-strand break in the helix. These enzymes are activated by sites on chromosomes where two double helices cross over each other such as those generated by supercoiling in front of a replication fork

Once a topoisomerase II molecule binds to such a crossing site, the protein uses ATP hydrolysis to perform the following set of reactions efficiently:

(1) it breaks one double helix reversibly to create a DNA “gate”;
(2) it causes the second, nearby double helix to pass through this opening; and
(3) it then reseals the break and dissociates from the DNA. At crossover points generated by supercoiling, passage of the double helix through the gate occurs in the direction that will reduce supercoiling. In this way, type II topoisomerases can relieve the overwinding tension generated in front of a replication fork. Their reaction mechanism also allows type II DNA topoisomerases to efficiently separate two interlocked DNA circles. Topoisomerase II also prevents the severe DNA tangling problems that would otherwise arise during DNA replication. This role is nicely illustrated by mutant yeast cells that produce, in place of the normal topoisomerase II, a version that is inactive above 37°C. When the mutant cells are warmed to this temperature, their daughter chromosomes remain intertwined after DNA replication and are unable to separate. The enormous usefulness of topoisomerase II for untangling chromosomes can readily be appreciated by anyone who has struggled to remove a tangle from a fishing line without the aid of scissors.