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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.
Each of the different cyclin-Cdk complexes serves as a molecular switch that triggers a specific cell-cycle event. We now consider how these switches initiate such events and how the cell-cycle control system ensures that the switches fire in the correct order and only once per cell cycle. We begin with the two central events of the cell cycle: the replication of DNA during S phase and the chromosome segregation and cell division of M phase. We then discuss how crucial regulatory mechanisms in G1 phase control whether or not a cell proliferates.
A cell must solve several problems in controlling the initiation and completion of DNA replication. Not only must replication occur with extreme accuracy to minimize the risk of mutations in the next cell generation, but every nucleotide in the genome must be copied once, and only once, to prevent the damaging effects of gene amplification. In Chapter 5, we discuss the sophisticated protein machinery that performs DNA replication with astonishing speed and accuracy. In this chapter, 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.
Early clues about the regulation of S phase came from studies in which human cells at various cell-cycle stages were fused to form single cells with two nuclei. These experiments revealed that when a G1 cell is fused with an S-phase cell, DNA replication occurs in the G1 nucleus (presumably triggered by S-Cdk activity in the S-phase cell). Fusion of a G2 cell with an S-phase cell, however, does not cause DNA synthesis in the G2 nucleus (Figure 17-21). These studies provided a clear hint that only G1 cells are competent to initiate DNA replication and that cells that have completed S phase (i.e. G2 cells) are not able to rereplicate their DNA, even when provided with S-Cdk activity. Apparently, passage through mitosis is required for the cell to regain the ability to undergo S phase.
Evidence from cell-fusion experiments for a rereplication block. These experiments were carried out in 1970 in cultured mammalian cells. (A) The results show that S-phase cytoplasm contains factors that drive a G1 nucleus directly into DNA synthesis. (more...)
We have begun to decipher the molecular basis of these cell fusion experiments only recently. DNA replication begins at origins of replication, which are scattered at various locations in the chromosome. Replication origins are simple and well defined in the budding yeast S. cerevisiae, and most of our understanding of the initiation machinery comes from studies of this organism. Analyses of proteins that bind to the yeast replication origin have identified a large, multiprotein complex known as the origin recognition complex (ORC). These complexes bind to replication origins throughout the cell cycle and serve as landing pads for several additional regulatory proteins.
One of these regulatory proteins is Cdc6. It is present at low levels during most of the cell cycle but increases transiently in early G1. It binds to ORC at replication origins in early G1, where it is required for the binding of a complex composed of a group of closely related proteins, the Mcm proteins. The resulting large protein complex formed at an origin is known as the pre-replicative complex, or pre-RC (Figure 17-22).
The initiation of DNA replication once per cell cycle. The ORC remains associated with a replication origin throughout the cell cycle. In early G1, Cdc6 associates with ORC. Aided by Cdc6, Mcm ring complexes then assemble on the adjacent DNA, resulting (more...)
Once the pre-RC has been assembled in G1, the replication origin is ready to fire. The activation of S-Cdk in late G1 pulls the trigger and initiates DNA replication. The initiation of replication also requires the activity of a second protein kinase, which collaborates with S-Cdk to cause the phosphorylation of ORC.
The S-Cdk not only initiates origin firing, but also helps to prevent rereplication in several ways. First, it causes the Cdc6 protein to dissociate from ORC after an origin has fired. This results in the disassembly of the pre-RC, which prevents replication from occurring again at the same origin. Second, it prevents the Cdc6 and Mcm proteins from reassembling at any origin. By phosphorylating Cdc6, it triggers Cdc6 ubiquitylation by the SCF enzyme complex discussed earlier. As a result, any Cdc6 protein that is not bound to an origin is rapidly degraded in proteasomes. S-Cdk also phosphorylates certain Mcm proteins, which triggers their export from the nucleus, further ensuring that the Mcm protein complex cannot bind to a replication origin (see Figure 17-22).
S-Cdk activity remains high during G2 and early mitosis, preventing rereplication from occurring after the completion of S phase. M-Cdk also helps ensure that rereplication does not occur during mitosis by phosphorylating the Cdc6 and Mcm proteins. The G1/S-Cdks help as well, by inducing Mcm export from the nucleus, ensuring that excess Mcm proteins that have not bound to origins in late G1 are taken out of action before replication begins.
Thus, several cyclin-Cdk complexes cooperate to restrain pre-RC assembly and prevent DNA rereplication after S phase. How, then, is the cell-cycle control system reset to allow replication to occur in the next cell cycle? The answer is simple. At the end of mitosis, all Cdk activity in the cell is reduced to zero. The resulting dephosphorylation of the Cdc6 and Mcm proteins allows pre-RC assembly to occur once again, readying the chromosomes for a new round of replication.
The completion of DNA replication leaves the G2 cell with two accurate copies of the entire genome, with each replicated chromosome consisting of two identical sister chromatids glued together along their length. The cell then undergoes the dramatic upheaval of M phase, in which the duplicated chromosomes and other cell contents are distributed equally to the two daughter cells. The events of mitosis are triggered by M-Cdk, which is activated after S phase is complete.
The activation of M-Cdk begins with the accumulation of M-cyclin (cyclin B in vertebrate cells, see Table 17-1). In embryonic cell cycles, the synthesis of M-cyclin is constant throughout the cell cycle, and M-cyclin accumulation results from a decrease in its degradation. In most cell types, however, M-cyclin synthesis increases during G2 and M, owing primarily to an increase in M-cyclin gene transcription. This increase in M-cyclin protein leads to a gradual accumulation of M-Cdk (the complex of Cdk1 and M-cyclin) as the cell approaches mitosis. Although the Cdk in these complexes is phosphorylated at an activating site by the enzyme CAK discussed earlier, it is held in an inactive state by inhibitory phosphorylation at two neighboring sites by the protein kinase Wee1 (see Figure 17-18). Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act, but the M-Cdk activity is repressed by the presence of two phosphate groups that block the active site of the kinase.
What, then, triggers the activation of the M-Cdk stockpile? The crucial event is the activation in late G2 of the protein phosphatase Cdc25, which removes the inhibitory phosphates that restrain M-Cdk (Figure 17-23). At the same time, the activity of the inhibitory kinase Wee1 is also suppressed, further ensuring that M-Cdk activity increases abruptly. Two protein kinases activate Cdc25. One, known as Polo kinase, phosphorylates Cdc25 at one set of sites. The other activating kinase is M-Cdk itself, which phosphorylates a different set of sites on Cdc25. M-Cdk also phosphorylates and inhibits Wee1.
The activation of M-Cdk. Cdk1 associates with M-cyclin as the levels of M-cyclin gradually rise. The resulting M-Cdk complex is phosphorylated on an activating site by the Cdk-activating kinase (CAK) and on a pair of inhibitory sites by the Wee1 kinase. (more...)
The ability of M-Cdk to activate its own activator (Cdc25) and inhibit its own inhibitor (Wee1) suggests that M-Cdk activation in mitosis involves a positive feedback loop (see Figure 17-23). According to this attractive model, the partial activation of Cdc25, perhaps by Polo kinase, leads to the partial activation of a subpopulation of M-Cdk complexes, which then phosphorylate more Cdc25 and Wee1 molecules. This leads to more M-Cdk dephosphorylation and activation, and so on. Such a mechanism would quickly promote the complete activation of all the M-Cdk complexes in the cell, converting a gradual increase in M-cyclin levels into a switchlike, abrupt rise in M-Cdk activity. As mentioned earlier, similar molecular switches operate at various points in the cell cycle to ensure that events such as entry into mitosis occur in an all-or-none fashion.
If a cell is driven into mitosis before it has finished replicating its DNA, it will pass on broken or incomplete sets of chromosomes to its daughter cells. This disaster is avoided in most cells by a DNA replication checkpoint mechanism, which ensures that the initiation of mitosis cannot occur until the last nucleotide in the genome has been copied. Sensor mechanisms, of unknown molecular nature, detect either the unreplicated DNA or the corresponding unfinished replication forks and send a negative signal to the cell-cycle control system, blocking the activation of M-Cdk. Thus, normal cells treated with chemical inhibitors of DNA synthesis, such as hydroxyurea, do not progress into mitosis. If the checkpoint mechanism is defective, however, as in yeast cells with certain mutations or in mammalian cells treated with high doses of caffeine, the cells plunge into a suicidal mitosis despite the failure to complete DNA replication (Figure 17-24).
The DNA replication checkpoint. In the experiments diagrammed here, mammalian cells in culture were treated with caffeine and hydroxyurea, either alone or in combination. Hydroxyurea blocks DNA synthesis. This block activates a checkpoint mechanism that (more...)
The final targets of the negative checkpoint signal are the enzymes that control M-Cdk activation. The negative signal activates a protein kinase that inhibits the Cdc25 protein phosphatase (see Figures 17-18 and 17-23). As a result, M-Cdk remains phosphorylated and inactive until DNA replication is complete.
One of the most remarkable features of cell-cycle control is that a single protein kinase, M-Cdk, is able to bring about all of the diverse and complex rearrangements that occur in the early stages of mitosis (discussed in Chapter 18). At a minimum, M-Cdk must induce the assembly of the mitotic spindle and ensure that replicated chromosomes attach to the spindle. In many organisms, M-Cdk also triggers chromosome condensation, nuclear envelope breakdown, actin cytoskeleton rearrangement, and the reorganization of the Golgi apparatus and endoplasmic reticulum. Each of these events is thought to be triggered when M-Cdk phosphorylates specific structural or regulatory proteins involved in the event, although most of these proteins have not yet been identified.
The breakdown of the nuclear envelope, for example, requires the disassembly of the nuclear lamina—the underlying shell of polymerized lamin filaments that gives the nuclear envelope its structural rigidity. Direct phosphorylation of lamin proteins by M-Cdk results in their depolymerization, which is an essential first step in the dismantling of the envelope (see Figure 12-21).
Chromosome condensation also seems to be a direct consequence of phosphorylation by M-Cdk. A complex of five proteins, known as the condensin complex, is required for chromosome condensation in Xenopus embryos. After M-Cdk has phosphorylated several subunits in the complex, two of the subunits are able to change the coiling of DNA molecules in a test tube. It is thought that this coiling activity is important for chromosome condensation during mitosis (see Figure 4-56).
Phosphorylation by M-Cdk also triggers the complex microtubule rearrangements and other events that lead to the assembly of the mitotic spindle. As discussed in Chapter 18, M-Cdk is known to phosphorylate a number of proteins that regulate microtubule behavior, causing the increase in microtubule instability that is required for spindle assembly.
After M-Cdk has triggered the complex rearrangements that occur in early mitosis, the cell cycle reaches its culmination with the separation of the sister chromatids at the metaphase-to-anaphase transition. Although M-Cdk activity sets the stage for this event, an entirely different enzyme complex—the anaphase-promoting complex (APC) introduced earlier—throws the switch that initiates sister-chromatid separation. The APC is a highly regulated ubiquitin ligase that promotes the destruction of several mitotic regulatory proteins (see Figure 17-20B).
The attachment of the two sister chromatids to opposite poles of the mitotic spindle early in mitosis results in forces tending to pull the two chromatids apart. These pulling forces are initially resisted because the sister chromatids are bound tightly together, both at their centromeres and all along their arms. This sister-chromatid cohesion depends on a complex of proteins, the cohesin complex, that is deposited along the chromosomes as they are duplicated in S phase. The cohesin proteins (cohesins) are closely related to the proteins of the condensin complex involved in chromosome condensation, suggesting a common evolutionary origin for the two processes (see Figure 18-3).
Anaphase begins with a sudden disruption of the cohesion between sister chromatids, which allows them to separate and move to opposite poles of the spindle. This process is initiated by a remarkable cascade of signaling events. The sister-chromatid separation requires the activation of the APC enzyme complex, suggesting that proteolysis is central to the process (Figure 17-25). The relevant target of the APC is the protein securin. Before anaphase, securin binds to and inhibits the activity of a protease called separase. The destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of the cohesin complex. In an instant, the cohesin complex falls away from the chromosomes, and the sister chromatids separate (Figure 17-26).
Two experiments that demonstrate the requirement for protein degradation to exit from mitosis. (A) An APC inhibitor was added to frog egg extracts undergoing mitosis in vitro (see Figure 17-9). The inhibitor arrested mitosis at metaphase, indicating that (more...)
The triggering of sister-chromatid separation by the APC. The activation of APC by Cdc20 leads to the ubiquitylation and destruction of securin, which normally holds separase in an inactive state. The destruction of securin allows separase to cleave a (more...)
If the APC triggers anaphase, what triggers the APC? The answer is only partly known. APC activation requires the protein Cdc20, which binds to and activates the APC at mitosis (see Figures 17-26 and 17-20B). At least two processes regulate Cdc20 and its association with the APC. First, Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene. Second, phosphorylation of the APC helps Cdc20 bind to the APC, thereby helping to create an active complex.
It is not clear what kinases phosphorylate and activate the Cdc20-APC complex. M-Cdk activity is required for the activity of these kinases, but there is a significant delay, or lag phase, between M-Cdk activation and the activation of the Cdc20-APC complex. The molecular basis of this delay is still mysterious, but it is likely to hold the key to how anaphase is initiated at the correct time in M phase.
The cell does not commit itself to the momentous events of anaphase before it is fully prepared. In most cell types, a spindle-attachment checkpoint mechanism operates to ensure that all chromosomes are properly attached to the spindle before sister-chromatid separation occurs. The checkpoint depends on a sensor mechanism that monitors the state of the kinetochore, the specialized region of the chromosome that attaches to microtubules of the spindle. Any kinetochore that is not properly attached to the spindle sends out a negative signal to the cell-cycle control system, blocking Cdc20-APC activation and sister-chromatid separation.
The nature of the signal generated by an unattached kinetochore is not clear, although several proteins, including Mad2, are recruited to unattached kinetochores and are required for the spindle-attachment checkpoint to function. Even a single unattached kinetochore in the cell results in Mad2 binding and the inhibition of Cdc20-APC activity and Securin destruction (Figure 17-27). Thus, sister-chromatid separation cannot occur until the last kinetochore is attached.
Mad2 protein on unattached kinetochores. This fluorescence micrograph shows a mammalian cell in prometaphase, with the mitotic spindle in green and the sister chromatids in blue. One sister chromatid pair is not yet attached to the spindle. The presence (more...)
Surprisingly, the normal timing of anaphase does not require a functional spindle-attachment checkpoint, at least in frog embryos and yeasts. Mutant yeast cells with a defective checkpoint undergo anaphase with normal timing, indicating that some other mechanism normally determines the timing of anaphase in these cells. In mammalian cells, however, a defect in the spindle-attachment checkpoint causes anaphase to occur slightly earlier than normal. This finding suggests that, in our cells, the checkpoint has evolved from a useful accessory to an essential component of the cell-cycle control system.
After the chromosomes have been segregated to the poles of the spindle, the cell must reverse the complex changes of early mitosis. The spindle must be disassembled, the chromosomes decondensed, and the nuclear envelope reformed. Because the phosphorylation of various proteins is responsible for getting cells into mitosis in the first place, it is not surprising that the dephosphorylation of these same proteins is required to get them out. In principle, these dephosphorylations and the exit from mitosis could be triggered by the inactivation of M-Cdk, the activation of phosphatases, or both. Evidence suggests that M-Cdk inactivation is primarily responsible.
M-Cdk inactivation occurs mainly by ubiquitin-dependent proteolysis of M-cyclins. Ubiquitylation of the cyclin is usually triggered by the same Cdc20-APC complex that promotes the destruction of Securin at the metaphase-to-anaphase transition (see Figure 17-20B). Thus, the activation of the Cdc20-APC complex leads not only to anaphase, but also to M-Cdk inactivation—which in turn leads to all of the other events that take the cell out of mitosis.
In early animal embryos, the inactivation of M-Cdk in late mitosis is due almost entirely to the action of Cdc20-APC. Recall, however, that M-Cdk stimulates Cdc20-APC activity (see Figure 17-26). Thus, the destruction of M-cyclin in late mitosis soon leads to the inactivation of all APC activity in an embryonic cell. This is a useful arrangement in rapid embryonic cell cycles, as APC inactivation immediately after mitosis allows the cell to quickly begin accumulating new M-cyclin for the next cycle (Figure 17-28A).
The creation of a G1 phase by stable Cdk inhibition after mitosis. (A) In early embryonic cell cycles, Cdc20-APC activity rises at the end of metaphase, triggering M-cyclin destruction. Because M-Cdk activity stimulates Cdc20-APC activity, the loss of (more...)
Rapid cyclin accumulation immediately after mitosis is not useful, however, in cell cycles containing a G1 phase. In these cycles, progression into the next S phase is delayed in G1 to allow for cell growth and for the cycle to be regulated by extracellular signals. Thus, most cells employ several mechanisms to ensure that Cdk reactivation is prevented after mitosis. One mechanism makes use of another APC-activating protein called Hct1, a close relative of Cdc20. Although both Hct1 and Cdc20 bind and activate the APC, they differ in one important respect. Whereas the Cdc20-APC complex is activated by M-Cdk, the Hct1-APC complex is inhibited by M-Cdk, which directly phosphorylates Hct1. As a result of this relationship, Hct1-APC activity increases in late mitosis after the Cdc20-APC complex has initiated the destruction of M-cyclin. M-cyclin destruction therefore continues after mitosis: although Cdc20-APC activity has declined, Hct1-APC activity is high (Figure 17-28B).
A second mechanism that suppresses Cdk activity in G1 depends on the increased production of CKIs, the Cdk inhibitory proteins discussed earlier. Budding yeast cells, in which this mechanism is best understood, contain a CKI protein called Sic1, which binds to and inactivates M-Cdk in late mitosis and G1. Like Hct1, Sic1 is inhibited by M-Cdk, which phosphorylates Sic1 during mitosis. M-Cdk also phosphorylates and inhibits a gene regulatory protein required for Sic1 synthesis, resulting in decreased Sic1 production. Thus, Sic1 and M-Cdk, like Hct1 and M-Cdk, mutually inhibit each other. As a result, the decline in M-Cdk activity that occurs in late mitosis triggers the rapid accumulation of Sic1 protein, and this CKI helps ensure that M-Cdk activity is stably inhibited after mitosis.
In most cells, M-Cdk inactivation in late mitosis also results from decreased transcription of M-cyclin genes. In budding yeast, for example, M-Cdk promotes the expression of these genes, resulting in a positive feedback loop. This loop is turned off as cells exit from mitosis: the inactivation of M-Cdk by Hct1 and Sic1 leads to decreased M-cyclin gene transcription and thus decreased M-cyclin synthesis.
In summary Hct1-APC activation, CKI accumulation, and decreased cyclin production act together to ensure that the early G1 phase is a time when essentially all Cdk activity is suppressed. As in many other aspects of cell-cycle control, the use of multiple regulatory mechanisms makes the suppression system robust, so that it still operates with reasonable efficiency even if one mechanism fails.
How does the cell escape from this stable G1 state to initiate S phase? As we describe later, escape usually occurs through the accumulation of G1-cyclins. In budding yeast, for example, these cyclins are not targeted for destruction by Hct1-APC and are not inhibited by Sic1. As a result, the accumulation of G1 cyclins leads to an unopposed increase in G1-Cdk activity (Figure 17-29). In animal cells, the accumulation of G1-cyclins is stimulated by the extracellular signals that promote cell proliferation, as we discuss later.
The control of G1 progression by Cdk activity in budding yeast. As cells exit from mitosis and inactivate M-Cdk, the resulting increase in Hct1 and Sic1 activities results in stable Cdk inactivation during G1. When conditions are right for entering a (more...)
In budding yeast, G1-Cdk activity triggers the transcription of G1/S-cyclin genes, leading to increased synthesis of G1/S-cyclins and the formation of G1/S-Cdk complexes, which are also resistant to Hct1-APC and Sic1. The increased G1/S-Cdk activity initiates the events that commit the cell to enter S phase. It stimulates the transcription of S-cyclin genes, leading to the synthesis of S-cyclins and the formation of S-Cdk complexes. These complexes are inhibited by Sic1, but G1/S-Cdk phosphorylates and inactivates Sic1, thereby causing S-Cdk activation. G1/S-Cdk and S-Cdk also phosphorylate and inactivate Hct1-APC. Thus, the same feedback loops that trigger rapid M-Cdk inactivation in late mitosis now work in reverse at the end of G1 to ensure the rapid and complete activation of S-Cdk activity.
The control of G1 progression and S-phase initiation is often disrupted in cancer cells, leading to unrestrained cell-cycle entry and cell proliferation (discussed in Chapter 23). To develop improved methods for controlling cancer growth, we need a better understanding of the proteins that control G1 progression in mammalian cells.
Animal cells suppress Cdk activity in G1 by the same three mechanisms mentioned earlier for budding yeast: Hct1 activation, the accumulation of a CKI protein (p27 in mammalian cells), and the inhibition of cyclin gene transcription. As in yeasts, the activation of G1-Cdk complexes reverses all three inhibitory mechanisms in late G1.
The best understood effects of G1-Cdk activity in animal cells are mediated by a gene regulatory protein called E2F. It binds to specific DNA sequences in the promoters of many genes that encode proteins required for S-phase entry, including G1/S-cyclins and S-cyclins. E2F function is controlled primarily by an interaction with the retinoblastoma protein (Rb), an inhibitor of cell-cycle progression. During G1, Rb binds to E2F and blocks the transcription of S-phase genes. When cells are stimulated to divide by extracellular signals, active G1-Cdk accumulates and phosphorylates Rb, reducing its affinity for E2F. The Rb then dissociates, allowing E2F to activate S-phase gene expression (Figure 17-30).
Mechanisms controlling S-phase initiation in animal cells. G1-Cdk activity (cyclin D-Cdk4) initiates Rb phosphorylation. This inactivates Rb, freeing E2F to activate the transcription of S-phase genes, including the genes for a G1/S-cyclin (cyclin E) (more...)
This transcriptional control system, like so many other control systems that regulate the cell cycle, includes feedback loops that sharpen the G1/S transition (see Figure 17-30):
As in yeast cells, the result of all these interactions is the rapid and complete activation of the S-Cdk complexes required for S-phase initiation.
The Rb protein was identified originally through studies of an inherited form of eye cancer in children, known as retinoblastoma (discussed in Chapter 23). The loss of both copies of the Rb gene leads to excessive cell proliferation in the immature retina, suggesting that the Rb protein is particularly important for restraining the rate of cell division in the developing retina. The complete loss of Rb does not immediately cause increased proliferation of other cell types, in part because Hct1 and p27 provide assistance in G1 control, and in part because other cell types contain Rb-related proteins that provide backup support in the absence of Rb. It is also likely that other proteins, unrelated to Rb, help to regulate the activity of E2F.
For proliferating cells to maintain a relatively constant size, the length of the cell cycle must match the time it takes the cell to double in size. If the cycle time is shorter than this, the cells will get smaller with each division; if it is longer, the cells will get bigger with each division. Because cell growth depends on nutrients and growth signals in the environment, the length of the cell cycle has to be able to adjust to varying environmental conditions (Figure 17-31). It is not clear how proliferating cells coordinate their growth with the rate of cell-cycle progression to maintain their size.
Cell size control through control of the cell cycle in yeasts. These graphs show the relationship between growth rate, cell size, and cell cycle time. (A) If cell division continued at an unchanged rate when cells were starved and stopped growing, the (more...)
There is evidence that budding yeasts coordinate their growth and cell-cycle progression by monitoring the total amount of a G1 cyclin called Cln3 (see Table 17-1, p. 994). Because Cln3 is synthesized in parallel with cell growth, its concentration remains constant while its total amount increases as the cell grows. If the amount of Cln3 is artificially increased, the cells divide at a smaller size than normal, whereas if it is artificially decreased, the cells divide at a larger size than normal. These experiments are consistent with the idea that the cells commit themselves to division when the total amount of Cln3 reaches some threshold value. How, then, can the cell monitor the total amount of Cln3, rather than its concentration? One possibility is that cells inherit a fixed amount of an inhibitor that can bind to Cln3 and block its activity. When the amount of Cln3 exceeds the amount of this inhibitor, the extra Cln3 triggers G1-Cdk activation and a new cell cycle. Since all cells receive a fixed and equal quantity of DNA, it has been speculated that the Cln3 inhibitor could be DNA itself, or some protein bound to DNA (Figure 17-32). Such a mechanism would also explain why cell size in all organisms is proportional to ploidy (the number of copies of the nuclear genome per cell).
A hypothetical model of how budding yeast cells might coordinate cell growth and cell-cycle progression. The cell contains a fixed number of proteins (red) that are bound to DNA and bind and inhibit Cln3 molecules (green). As the cell grows, the total (more...)
Whereas yeast cells grow and proliferate constitutively if nutrients are plentiful, animal cells generally grow and proliferate only when they are stimulated to do so by signals from other cells. The size at which an animal cell divides depends, at least in part, on these extracellular signals, which can regulate cell growth and proliferation independently. Animal cells can also completely uncouple cell growth and division so as to grow without dividing or to divide without growing. The eggs of many animals, for example, grow to an extremely large size without dividing. After fertilization, this relationship is reversed, and many rounds of division occur without growth (see Figure 17-8). Thus, although cell growth and cell division are usually coordinated, they can be regulated independently. Cell growth does not depend on cell-cycle progression. Yeast cells continue to grow when cell-cycle progression is blocked by a mutation; and many animal cells, including neurons and muscle cells, grow large after they have withdrawn permanently from the cell cycle.
When chromosomes are damaged, as can occur after exposure to radiation or certain chemicals, it is essential that they be repaired before the cell attempts to duplicate or segregate them. The cell-cycle control system can readily detect DNA damage and arrest the cycle at DNA damage checkpoints. Most cells have at least two such checkpoints—one in late G1, which prevents entry into S phase, and one in late G2, which prevents entry into mitosis.
The G2 checkpoint depends on a mechanism similar to the one discussed earlier that delays entry into mitosis in response to incomplete DNA replication. When cells in G2 are exposed to damaging radiation, for example, the damaged DNA sends a signal to a series of protein kinases that phosphorylate and inactivate the phosphatase Cdc25. This blocks the dephosphorylation and activation of M-Cdk, thereby blocking entry into mitosis. When the DNA damage is repaired, the inhibitory signal is turned off, and cell-cycle progression resumes.
The G1 checkpoint blocks progression into S phase by inhibiting the activation of G1/S-Cdk and S-Cdk complexes. In mammalian cells, for example, DNA damage leads to the activation of the gene regulatory protein p53, which stimulates the transcription of several genes. One of these genes encodes a CKI protein called p21, which binds to G1/S-Cdk and S-Cdk and inhibits their activities, thereby helping to block entry into S phase.
DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations. This is because it interacts with another protein, Mdm2, that acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. DNA damage activates protein kinases that phosphorylate p53 and thereby reduce its binding to Mdm2. This decreases p53 degradation, which results in a marked increase in p53 concentration in the cell. In addition, the decreased binding to Mdm2 enhances the ability of p53 to stimulate gene transcription (Figure 17-33).
How DNA damage arrests the cell cycle in G1. When DNA is damaged, protein kinases that phosphorylate p53 are activated. Mdm2 normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. Phosphorylation of p53 blocks its binding (more...)
Like many other checkpoints, DNA damage checkpoints are not essential for normal cell division if environmental conditions are ideal. Conditions are rarely ideal, however: a low level of DNA damage occurs in the normal life of any cell, and this damage accumulates in the cell's progeny if the damage checkpoints are not functioning. Over the long term, the accumulation of genetic damage in cells lacking checkpoints leads to an increased frequency of cancer-promoting mutations. Indeed, mutations in the p53 gene occur in at least half of all human cancers (discussed in Chapter 23). This loss of p53 function allows the cancer cell to accumulate mutations more readily. Similarly, a rare genetic disease known as ataxia telangiectasia is caused by a defect in one of the protein kinases that phosphorylates and activates p53 in response to x-ray-induced DNA damage; patients with this disease are very sensitive to x-rays due to the loss of the DNA damage checkpoints, and they consequently suffer from increased rates of cancer.
What if DNA damage is so severe that repair is not possible? In this case, the response is different in different organisms. Unicellular organisms such as budding yeast transiently arrest their cell cycle to repair the damage. If repair cannot be completed, the cycle resumes despite any damage. For a single-celled organism, life with mutations is apparently better than no life at all. In multicellular organisms, however, the health of the organism takes precedence over the life of an individual cell. Cells that divide with severe DNA damage threaten the life of the organism, since genetic damage can often lead to cancer and other lethal defects. Thus, animal cells with severe DNA damage do not attempt to continue division, but instead commit suicide by undergoing programmed cell death, or apoptosis, as we discuss in the next section. The decision to die in this way also depends on the activation of p53, and it is this function of p53 that is apparently most important in protecting us against cancer.
As a review, the major cell-cycle regulatory proteins are summarized in Table 17-2, with the general structure of the cell-cycle control system shown in Figure 17-34.
Summary of the Major Cell-cycle Regulatory Proteins.
An overview of the cell-cycle control system. The core of the cell-cycle control system consists of a series of cyclin-Cdk complexes (yellow). The activity of each complex is also influenced by various inhibitory checkpoint mechanisms, which provide information (more...)
An ordered sequence of cyclin-Cdk activities triggers most of the events of the cell cycle. During G1 phase, Cdk activity is reduced to a minimum by Cdk inhibitors (CKIs), cyclin proteolysis, and decreased cyclin gene transcription. When environmental conditions are favorable, G1- and G1/S-Cdks increase in concentration, overcoming these inhibitory barriers in late G1 and triggering the activation of S-Cdk. The S-Cdk phosphorylates proteins at DNA replication origins, initiating DNA synthesis through a mechanism that ensures that the DNA is duplicated only once per cell cycle.
Once S phase is completed, the activation of M-Cdk leads to the events of early mitosis, whereby the cell assembles a mitotic spindle and prepares for segregation of the duplicated chromosomes—which consist of sister chromatids glued together. Anaphase is triggered by the destruction of the proteins that hold the sisters together. The M-Cdk is then inactivated by cyclin proteolysis, which leads to cytokinesis and the end of M phase. Progression through the cell cycle is regulated precisely by various inhibitory mechanisms that arrest the cell cycle at specific checkpoints when events are not completed successfully, when DNA damage occurs, or when extracellular conditions are unfavorable.
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