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MOLECULAR CELL BIOLOGY SEVENTH EDITION

CHAPTER 19 The Eukaryotic Cell Cycle

Copyright © 2013 by W. H. Freeman and Company

Figure 19.1 The fate of a single parental chromosome throughout the eukaryotic cell cycle.

The fate of a single parental chromosome throughout the eukaryotic cell cycle 1. The cell cycle entails an ordered series of macromolecular events that lead to cell division and the production of two daughter cells each containing chromosomes identical to those of the parental cell. As illustrated in Figure 19-1, the cell cycle is divided into four major phases. In cycling (replicating) somatic cells, cells synthesize RNAs and proteins during the G1 phase, preparing for DNA synthesis and chromosome replication during the S (synthesis) phase. After progressing through the G2 phase, cells begin the complicated process of mitosis, also called the M (mitotic) phase, which is divided into several stages. In multicellular organisms, most differentiated cells “exit” the cell cycle and survive for days, weeks, or in some cases (e.g., nerve cells and cells of the eye lens) even the lifetime of the organism without dividing again. Such postmitotic cells generally exit the cell cycle in G1, entering a phase called G0 (Figure 19-1). Some G0 cells can return to the cell cycle and resume replicating; this reentry is regulated, thereby providing control of cell proliferation. 2. In vertebrates and diploid yeasts, cells in G1 have a diploid number of chromosomes (2n), one inherited from each parent. In haploid yeasts, cells in G1 have one of each chromosome (1n), the haploid number. Rapidly replicating human cells progress through the full cell cycle in about 24 hours: mitosis takes ≈30 minutes; G1, 9 hours; the S phase, 10 hours; and G2, 4.5 hours.

Figure 19.3 The budding yeast S. cerevisiae.

Figure 19.4 The fission yeast S. pombe.

Experimental Figure 19.5 Wild-type cell division cycle (CDC) genes can be isolated from a S. cerevisiae genomic library by functional complementation of cdc mutants.

Genetic Studies with S. cerevisiae and S. pombe 1. S. cerevisiae cells replicate by budding (Figure 19-3). Both mother and daughter cells remain in the G1

period of the cell cycle while growing, although it takes the initially larger mother cells a shorter time to reach a size compatible with cell division. When S. cerevisiae cells in G1 have grown sufficiently, they begin a program of gene expression that leads to entry into the S phase. If G1 cells are shifted from a rich medium to a medium low in nutrients before they reach a critical size, they remain in G1 and grow slowly until they are large enough to enter the S phase. However, once G1 cells reach the critical size, they become committed to completing the cell cycle, entering the S phase and proceeding through G2 and mitosis, even if they are shifted to a medium low in nutrients. The point in late G1 of growing S. cerevisiae cells when they become irrevocably committed to entering the S phase and traversing the cell cycle is called START.

2. Identification of the catalytic protein kinase subunit of MPF and further insight into its regulation came from genetic analysis of the cell cycle in the fission yeast S. pombe. This yeast grows as a rod-shaped cell that increases in length as it grows and then divides in the middle during mitosis to produce two daughter cells of equal size (Figure 19-4).

3. The phenotypic behavior of temperature-sensitive cdc28 mutants indicates that Cdc28 function is critical for entry into the S phase. When these mutants are shifted to the nonpermissive temperature, they behave like wild-type cells suddenly deprived of nutrients. That is, cdc28 mutant cells that have grown large enough to pass START at the time of the temperature shift continue through the cell cycle normally and undergo mitosis, whereas those that are too small to have passed START when shifted to the nonpermissive temperature do not enter the S phase even though nutrients are plentiful. Even though cdc28 cells blocked in G1 continue to grow in size at the nonpermissive temperature, they cannot pass START and enter the S phase. Thus they appear as large cells with no bud. The wild-type CDC28 gene was isolated by its ability to complement mutant cdc28 cells at the nonpermissive temperature. Note that in S. cerevisiae wild-type genes are indicated in italic capital letters (e.g., CDC28) and recessive mutant genes in italic lowercase letters (e.g., cdc28); the corresponding wild-type protein is written in Roman letters with an initial capital (e.g., Cdc28), similar to S. pombe proteins. Each type of yeast contains a single cyclin-dependent protein kinase (CDK), which can substitute for each other: Cdc2 in S. pombe and Cdc28 in S. cerevisiae

Figure 17-14 Molecular Biology of the Cell (© Garland Science 2008)

Checkpoints in the cell-cycle control system. In most cells there are several points in the cell cycle, called checkpoints, at which the cycle can be arrested if previous events have not been completed. Entry into mitosis is prevented, for example, when DNA replication is not complete, and chromosome separation in mitosis is delayed if some chromosomes are not properly attached to the mitotic spindle. Progression through G1 and G2 is delayed by braking mechanisms if the DNA in the chromosomes is damaged by radiation or chemicals. Delays at these DNA damage checkpoints provide time for the damaged DNA to be repaired, after which the cell-cycle brakes are released and progress resumes. Checkpoints are important in another way as well. They are points in the cell cycle at which the control system can be regulated by extracellular signals from other cells. These signals—which can either promote or inhibit cell proliferation—tend to act by regulating progression through a G1 checkpoint, using mechanisms discussed later in the chapter. (Figure 17-14 Molecular Biology of the Cell - http://www.ncbi.nlm.nih.gov/books/NBK26824/)

Figure 19.10 Regulation of cell cycle transitions.

Regulation of cell cycle transitions Figure 19-10 outlines the role of the three major classes of cyclin-CDK complexes that control passage through the cell cycle: the G1, S-phase, and mitotic cyclin-CDK complexes. 1. When cells are stimulated to replicate, G1 cyclin-CDK complexes are expressed first. These prepare

the cell for the S phase by activating transcription factors that promote transcription of genes encoding enzymes required for DNA synthesis and the genes encoding S-phase cyclins and CDKs. The activity of S-phase cyclin-CDK complexes is initially held in check by inhibitors. Late in G1, the G1 cyclin-CDK complexes induce degradation of the S-phase inhibitors by phosphorylating them and consequently stimulating their polyubiquitination by the multiprotein SCF ubiquitin ligase. Subsequent degradation of the polyubiquitinated S-phase inhibitor by proteasomes releases active S-phase cyclin-CDK complexes.

2. Once activated, the S-phase cyclin-CDK complexes phosphorylate regulatory sites in the proteins that form DNA pre-replication complexes, which are assembled on replication origins during G1. Phosphorylation of these proteins not only activates initiation of DNA replication but also prevents reassembly of new pre-replication complexes.

3. Mitotic cyclin-CDK complexes are synthesized during the S phase and G2, but their activities are held in check by phosphorylation at inhibitory sites until DNA synthesis is completed. Once activated by dephosphorylation of the inhibitory sites, mitotic cyclin-CDK complexes phosphorylate multiple proteins that promote chromosome condensation, retraction of the nuclear envelope, assembly of the mitotic spindle apparatus, and alignment of condensed chromosomes at the metaphase plate. During mitosis, the anaphasepromoting complex (APC), a multisubunit ubiquitin ligase, polyubiquitinates key regulatory proteins marking them for proteasomal degradation. Once all the chromosomes are aligned, the APC polyubiquitinates securin, leading to its proteasomal degradation and the subsequent degradation of the cross-linking proteins connecting sister chromatids. This sequence of events initiates anaphase by freeing sister chromatids to segregate to opposite spindle poles. Late in anaphase, the APC also directs polyubiquitination and subsequent proteasomal degradation of the mitotic cyclins. As a result, the now separated chromosomes decondense, the nuclear envelope re-forms around daughter-cell nuclei, and the Golgi apparatus reassembles during telophase; finally, the cytoplasm divides at cytokinesis, yielding the two daughter cells.

Figure 19.11 An overview of how CDKs regulate cell cycle progression.

Regulation of CDK activity 1. The concentrations of the cyclins, the regulatory subunits of the heterodimeric protein kinases that control cell-cycle events, increase and decrease as cells progress through the cell cycle. The catalytic subunits of these kinases, called cyclin-dependent kinases (CDKs), have no kinase activity unless they are associated with a cyclin. Each CDK can associate with different cyclins, and the associated cyclin determines which proteins are phosphorylated by a particular cyclin-CDK complex (Figure 17-15 Molecular Biology of the Cell). 2. A key discovery in cell cycle studies was that cyclin-dependent kinases govern progression through the cell cycle. Three key features about these kinases are important to keep in mind throughout this chapter: ① Cyclin-dependent kinases (CDKs) are only active when bound to a regulatory cyclin subunit. ② Different types of cyclin-CDK complexes initiate different events. G1 CDKs and G1/S phase CDKs

promote entry into the cell cycle, S phase CDKs trigger S phase, and mitotic CDKs initiate the events of mitosis (Figure 19-11).

③ Multiple mechanisms are in place to ensure that the different CDKs are only active in the stages of the cell cycle they trigger.

Figure 19.12 Structural models of human CDK2.

Structural models of human CDK2 Unlike both fission and budding yeasts, each of which produce just one CDK, vertebrates produce several CDKs. The three-dimensional structure of one human cyclin-dependent kinase (CDK2) has been determined and provides insight into how cyclin binding and phosphorylation of CDKs regulate their protein kinase activity. ① Unphosphorylated, inactive CDK2 contains a flexible region, called the T-loop, that blocks access of

protein substrates to the active site where ATP is bound (Figure 19-12a). ② Unphosphorylated CDK2 bound to one of its cyclin partners, cyclin A, has minimal but detectable

protein kinase activity in vitro, although it may be essentially inactive in vivo. Extensive interactions between cyclin A and the T-loop cause a dramatic shift in the position of the T-loop, thereby exposing the CDK2 active site (Figure 19-12b).

③ Binding of cyclin A also shifts the position of the 1 helix in CDK2, modifying its substrate-binding surface. Phosphorylation of the activating threonine, located in the T-loop, causes additional conformational changes in the cyclin A–CDK2 complex that greatly increase its affinity for protein substrates (Figure 19-12c).

Experimental Figure 19.13 Mitotic cyclins are rate limiting for mitosis.

Mitotic cyclins are rate limiting for mitosis In all cases, MPF activity and cyclin B concentration were determined at various times after addition of sperm nuclei to a Xenopus egg extract treated as indicated in each panel. 1. These studies showed that MPF activity rises and falls in synchrony with the concentration of cyclin B (Figure 19-

13a). The early events of mitosis—chromosome condensation and nuclear envelope disassembly—occurred when MPF activity reached its highest levels in parallel with the rise in cyclin B concentration. Addition of cycloheximide, an inhibitor of protein synthesis, prevented cyclin B synthesis and also prevented the rise in MPF activity, chromosome condensation, and nuclear envelope disassembly.

2. To test the functions of cyclin B in these cell-cycle events, all mRNAs in the egg extract were degraded by digestion with a low concentration of RNase, which then was inactivated by addition of a specific inhibitor. This treatment destroys mRNAs without affecting the tRNAs and rRNAs required for protein synthesis, since their degradation requires much higher concentrations of RNase. When sperm nuclei were added to the RNase-treated extracts, the 1n nuclei replicated their DNA, but the increase in MPF activity and the early mitotic events (chromosome condensation and nuclear envelope disassembly), which the untreated extract supports, did not occur (Figure 19-13b).

3. Addition of cyclin B mRNA, produced in vitro from cloned cyclin B cDNA, to the RNase-treated egg extract and sperm nuclei restored the parallel oscillations in the cyclin B concentration and MPF activity and the characteristic early and late mitotic events as observed with the untreated egg extract (Figure 19-13c). Since cyclin B is the only protein synthesized under these conditions, these results demonstrate that it is the crucial protein whose synthesis is required to regulate MPF activity and the cycles of chromosome condensation and nuclear envelope breakdown mediated by cycling Xenopus egg extracts.

4. In these experiments, chromosome decondensation and nuclear envelope assembly (late mitotic events) coincided with decreases in the cyclin B level and MPF activity. To determine whether degradation of cyclin B is required for exit from mitosis, researchers added a mutant mRNA encoding a nondegradable cyclin B to a mixture of RNase-treated Xenopus egg extract and sperm nuclei. As shown in Figure 19-13d, MPF activity increased in parallel with the level of the mutant cyclin B, triggering condensation of the sperm chromatin and nuclear envelope disassembly (early mitotic events). However, the mutant cyclin B produced in this reaction never was degraded. As a consequence, MPF activity remained elevated, and the late mitotic events of chromosome decondensation and nuclear envelope formation were both blocked. This experiment demonstrates that the fall in MPF activity and exit from mitosis depend on degradation of cyclin B. The results of the two experiments with RNase-treated extracts show that entry into mitosis requires the accumulation

Cyclins determine the activity of CDKs 1. Cyclins are so named because their levels change during the cell cycle. They form a family of proteins that is defined by three key features: ① Cyclins bind to and activate CDKs. The activity and substrate specificity of any given CDK is primarily

defined by the particular cyclin to which it is bound. ② Cyclins are only present during the cell cycle stage that they trigger and are absent in other cell cycle

stage. ③ Cyclins not only regulate a particular cell cycle stage but also set in motion a series of events in

preparation for the next cell cycle stage. In this way, they propel the cell cycle forword. 2. Cyclins are divided into four classes defined by their presence and activity during the cell cycle: G1 cyclins, G1/S cyclins, S phase cyclins, and mitotic cyclins (Table 19-1).

The activation of M-Cdk The activation of M-Cdk begins with the accumulation of M-cyclin (cyclin B in vertebrate cells). 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 Wee 1. 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. 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. (Figure 17-25 Molecular Biology of the Cell - http://www.ncbi.nlm.nih.gov/books/NBK26856/#A3223)


CDK inhibitory protein (CKI)

CDK inhibitory protein (CKI) Cyclin-Cdk complexes can also be regulated by the binding of Cdk inhibitor proteins (CKIs). There are a variety of CKI proteins, and they are primarily employed in the control of G1 and S phase. The three-dimensional structure of a cyclin-Cdk-CKI complex reveals that CKI binding dramatically rearranges the structure of the Cdk active site, rendering it inactive (Figure 17-19). The final layer of control that is of proteins known as CDK inhibitors, or CKIs, that directly bind to the cyclin-CDK complex and inhibit its activity. As we will see in Section 19.4, these proteins play an especially important role in the regulation of the G1-S phase transition and its integration with extracellular signals. It thus comes as no surprise that the genes encoding these CKIs are often found mutated in human cancers. (Figure 17-19 Molecular Biology of the Cell - http://www.ncbi.nlm.nih.gov/books/NBK26824/#A3207)

Regulators of Cyclin-CDK activity Multiple mechanisms ensure that CDKs are active in the right stage of the cell cycle. Table 19-2 lists these key regulators of CDKs. Four proteins regulate the protein kinase activity of the S. pombe CDK. First is Cdc13, the mitotic cyclin of S. pombe (equivalent to cyclin B in metazoans), which associates with the CDK to form MPF with extremely low activity. 1. Kinases and phosphatases ① Second is the Wee1 protein-tyrosine kinase, which phosphorylates an inhibitory tyrosine residue (Y15)

in the CDK subunit. ② Third is another kinase, designated CDK-activating kinase (CAK), which phosphorylates an activating

threonine residue (T161). When both residues are phosphorylated, MPF is inactive. ③ Finally, the Cdc25 phosphatase removes the phosphate from Y15, yielding highly active MPF. 2. Inhibitory proteins ① As the S-phase cyclin-CDK heterodimers accumulate in late G1, they are immediately inactivated by

binding of an inhibitor, called Sic1, that is expressed late in mitosis and in early G1. Because Sic1 specifically inhibits B-type cyclin-CDK complexes, but has no effect on the G1 cyclin-CDK complexes, it functions as an S-phase inhibitor.

② All eukaryotes habor CKIs involved in regulating S phase and mitotic CDKs. Although these inhibitors display little sequence similarity, they are all essential to prevent premature activation of S phase and M phase CDKs. Inhibitors of G1 CDKs play an essential role in mediating a G1 arrest in response to proliferation inhibitory signals. A three proteins-p21cip, p27kip1, and p57kip2. These CKIs and must be degraded before DNA replication can begin.

③ A class of CKIs called INK4s (inhibitors of kinase 4) includes several small, closely related proteins that interact only with the G1 CDKs. Binding of INK4s to CDK4 and CDK6 blocks their interaction with cyclin D and hence their protein kinase activity.

Figure 19.30 Fundamental processes in the eukaryotic cell cycle.

Mitotic CDK (mitosis-promoting factor (MPF), CDK1/cycinB)

MPF 1) chromosome condensation 2) nuclear envelope breakdown 3) chromosome segregation

Mitotic CDK (mitosis-promoting factor (MPF), CDK1/cycinB) A heterodimer composed of a mitotic cyclin and CDK is commonly referred to as a mitosis-promoting factor (MPF). As we will see shortly, MPF turned out to be the key factor that regulates the initiation of mitosis in all eukaryotic cells. Presumably, the entry into mitosis is a consequence of the phosphorylation of specific proteins by the protein kinase activity of MPF. Although many of the critical substrates of MPF remain to be identified, we now know of examples that show how regulation by MPF phosphorylation mediates many of the early events of mitosis leading to metaphase: chromosome condensation, formation of the mitotic spindle, and disassembly of the nuclear envelope. Recall that a decrease in mitotic cyclins and the associated inactivation of MPF coincides with the later stages of mitosis. Just before this, in early anaphase, sister chromatids separate and move to opposite spindle poles. During telophase, microtubule dynamics return to interphase conditions, the chromosomes decondense, the nuclear envelope re-forms, the Golgi complex is remodeled, and cytokinesis occurs. Some of these processes are triggered by dephosphorylation; others, by protein degradation.

Figure 19.22 The nuclear lamina and its regulation by phosphorylation.

Figure 19.23 Nuclear envelope proteins phosphorylated by mitotic CDKs.

Figure 19.22 The nuclear lamina and its regulation by phosphorylation.

Figure 19.23 Nuclear envelope proteins phosphorylated by mitotic CDKs.

Nuclear envelope breakdown The nuclear envelope is a double-membrane extension of the rough endoplasmic reticulum containing many nuclear pore complexes. The lipid bilayer of the inner nuclear membrane is supported by the nuclear lamina, a meshwork of lamin filaments located adjacent to the inside face of the nuclear envelope (Figure 19-22a). The three nuclear lamins (A, B, and C) present in vertebrate cells belong to the class of cytoskeletal proteins, the intermediate filaments, that are critical in supporting cellular membranes. All three nuclear lamins form dimers containing a rodlike α-helical coiled-coil central section and globular head and tail domains; polymerization of these dimers through head-to-head and tail-to-tail associations generates the intermediate filaments that compose the nuclear lamina.

1. Early in mitosis, MPF phosphorylates specific serine residues in all three nuclear lamins, causing

depolymerization of the lamin intermediate filaments (Figure 19-22b). The phosphorylated lamin A and C dimers are released into solution, whereas the phosphorylated lamin B dimers remain associated with the nuclear membrane via their isoprenyl anchor. Depolymerization of the nuclear lamins leads to disintegration of the nuclear lamina meshwork and contributes to disassembly of the nuclear envelope. Disassembly of the nuclear envelope, which normally occurs early in mitosis, depends on phosphorylation of lamin A.

2. In addition, MPF-catalyzed phosphorylation of specific nucleoporins causes nuclear pore complexes to dissociate into subcomplexes during prophase. Similarly, phosphorylation of integral membrane proteins of the inner nuclear membrane is thought to decrease their affinity for chromatin and contribute to disassembly of the nuclear envelope. The weakening of the associations between the inner nuclear membrane and the nuclear lamina and chromatin may allow sheets of inner nuclear membrane to retract into the endoplasmic reticulum, which is continuous with the outer nuclear membrane (Figure 19-23).

Figure 19.2 The stages of mitosis.

Metaphase-to-anaphase transition


Figure 19.27 Regulation of cohesin cleavage.

Chromosome segregation 1. 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. As mentioned earlier, each sister chromatid of a metaphase chromosome is attached to microtubules via its kinetochore, a complex of proteins assembled at the centromere. The opposite ends of these kinetochore microtubules associate with one of the spindle poles. At metaphase, the spindle is in a state of tension with forces pulling the two kinetochores toward the opposite spindle poles balanced by forces pushing the spindle poles apart. Sister chromatids do not separate because they are held together at their centromeres and multiple positions along the chromosome arms by multiprotein complexes called cohesins. Among the proteins composing the cohesin complexes are members of the SMC protein family. 2. Recent genetic studies in the budding yeast S. cerevisiae have led to the model depicted in Figure 19-27 for how the APC regulates sister chromatid separation to initiate anaphase. Cohesin SMC proteins bind to each sister chromatid; other subunits of cohesin, including Scc1, then link the SMC proteins, firmly associating the two chromatids. The cross-linking activity of cohesin depends on securin, which is found in all eukaryotes. Prior to anaphase, securin binds to and inhibits separase, a ubiquitous protease related to the caspase proteases that regulate programmed cell death. Once all chromosome kinetochores have attached to spindle microtubules, the APC is directed by a specificity factor called Cdc20 to polyubiquitinate securin, leading to the onset of anaphase. (This specificity factor is distinct from Cdh1, which directs the APC to polyubiquitinate B-type cyclins.) Polyubiquitinated securin is rapidly degraded by proteasomes, thereby releasing separase. Free from its inhibitor, separase cleaves Scc1, breaking the protein cross-link between sister chromatids. Once this link is broken, the poleward force exerted on kinetochores can move sister chromatids toward the opposite spindle poles. 3. Because Cdc20—the specificity factor that directs APC to securin—is activated before Cdh1—the specificity factor that directs APC to mitotic cyclins—MPF activity does not decrease until after the chromosomes have segregated. As a result of this temporal order in the activation of the two APC specificity factors (Cdc20 and Cdh1), the chromosomes remain in the condensed state and reassembly of the nuclear envelope does not occur until chromosomes are moved to the proper position (Figure 17-44 Molecular Biology of the Cell).

Anaphase-promoting complex/Cyclosome (APC/C)

Nat Rev Cancer (2006) 6(5):369-381

Anaphase-promoting complex/Cyclosome (APC/C) 1. Levels of cyclin expression during cell division are periodic. This is the result of a constant synthetic rate coupled with a defined window in the cycle of specific proteolysis, which is executed by the ubiquitin proteasome system (UPS). CKIs, negative-regulators of cyclin–CDK kinase complexes, are also targeted for degradation by the UPS. Therefore, the cell cycle is predominantly regulated by two types of post-translational protein modification — phosphorylation and ubiquitylation. The UPS comprises two discrete steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and degradation of the polyubiquitylated protein by the 26S proteasome complex3 (FIG. 1). The first step is mediated by at least three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligase (E3). An involvement of E1 in cancer has not been described, and only a few reports have linked E2 to cancer development4. By contrast, a large amount of evidence indicates that deregulation of E3 ligase, which is involved in many biological systems, often results in cancer development. 2. Overview of the ubiquitin-proteasome pathway. Ubiquitin (Ub) is a small 8-kDa protein, which is first transferred to the ubiquitin-activating enzyme, E1, in an ATPdependent manner. This activated ubiquitin is then transferred to the ubiquitin conjugating enzyme, E2. Finally, the ubiquitin is covalently attached to the target protein by E3 ubiquitin ligase, leading to the formation of a polyubiquitin chain. The polyubiquitylated protein is recognized by the 26S proteasome, and is destroyed in an ATP-dependent manner. There are many E3 ubiquitin ligases, which are categorized into four major classes: HECT-type, RING-finger-type, U-box-type and PHD-finger-type. RING-finger-type E3s are further divided into subfamilies, including cullin-based E3s, which constitute one of the largest classes of E3s. There are seven cullin-based E3s including the SKP1–CUL1–F-box-protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C) (Nat Rev Cancer (2006) 6(5):369-381).

Nat Rev Cancer (2006) 6(5):369-381

The structural similarity of SCF and APC/C SCF and APC/C: similar but distinct. E3 components in the UPS are thought to be primarily responsible for the specific recognition of a large number of target proteins. This requires both specificity and versatility, which are provided by the existence of 500–1,000 different E3 ligases. These are now categorized into four major classes on the basis of their specific structural motif: HECT-type, RING finger-type, U-box-type or PHD-finger-type. RING-finger-type E3s are thought to be the largest family and are further divided into subfamilies; one of these, the cullin-based E3 subfamily, is one of the largest single classes of E3. There are seven cullin-based E3s, including the SKP1–CUL1–F-box-protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C), both of which are involved in the proteolysis of core components of the cell-cycle machinery. The SCF complex consists of three invariable components — RBX1 (RING-finger protein), CUL1 (scaffold protein), and SKP1 (adaptor protein) — as well as one variable component, known as an F-box protein, that binds through its F-box motif to SKP1 and is responsible for substrate recognition (FIG. 2). The APC/C consists of the invariable core components APC1 (RBX1-related RING-finger protein), APC2 (CUL1-related scaffold protein) and at least 11 other components, the functions of which are not fully understood. In addition, there is the variable activator subunit, cell division cycle 20 (CDC20) or CDH1 (also known as HCT1), both of which confer substrate specificity in the same way as F-box proteins do in the SCF complex. Known substrates of each variable subunit in SCF and APC/C are shown (Nat Rev Cancer (2006) 6(5):369-381)

APC/C-CDC20/proteasome degrades securin APC/C-CDC20 contributes to the proteolysis of securin, and thereby triggers chromosomal separation at anaphase. After replication, sister chromatids are accompanied until anaphase by the multiprotein complex cohesin. Separase cleaves cohesin, but its activity is suppressed by securin. The degradation of securin by APC/C-CDC20 activates separase, resulting in the cleavage of cohesin and separation of sister chromatids (Figure 19-30, Nat Rev Cancer (2006) 6(5):369-381). Because Cdc20—the specificity factor that directs APC to securin—is activated before Cdh1—the specificity factor that directs APC to mitotic cyclins—MPF activity does not decrease until after the chromosomes have segregated (Figure 19-30). As a result of this temporal order in the activation of the two APC specificity factors (Cdc20 and Cdh1), the chromosomes remain in the condensed state and reassembly of the nuclear envelope does not occur until chromosomes are moved to the proper position.

Cyclin B should be degraded for 1) chromosome decondensation 2) reformation of interphase MT array chromosome decondensation

MPF

Nat Rev Cancer (2006) 6(5):369-381

APC/C-Cdh1/proteasome degrades mitotic cyclins 1. We saw earlier that in late anaphase, polyubiquitination of mitotic cyclin by the anaphase-promoting complex (APC) leads to the proteasomal destruction of this cyclin. Additional experiments with Xenopus egg extracts provided evidence that degradation of cyclin B, the Xenopus mitotic cyclin, and the resulting decrease in MPF activity are required for chromosome decondensation but not for chromosome segregation. The fall in MPF activity in telophase allows constitutive protein phosphatases to remove the regulatory phosphates from condensin, lamins, nucleoporins, and other nuclear membrane proteins, permitting the decondensation of chromosomes and the reassembly of the nuclear membrane, nuclear lamina, and nuclear pore complexes. When mitosis is complete, the spindle disassembles and the interphase microtubule network re-forms (Figure 18-34a part 2 6th ed). 2. The APC/C is structurally similar to the SCF complex, and consists of invariable core components — APC11 (RBX1-related RING-finger protein), APC2 (CUL1-related scaffold protein) and at least 11 other components without a defined role — as well as a variable component known as an activator. There are two such variable components in mitotically cycling cells — cell division cycle 20 (CDC20) and CDH1 (also known as HCT1)— and they confer substrate specificity in the same way that F-box proteins do in the SCF complex. In late M phase, CDC20 is ubiquitylated, and therefore degraded, by APC/C-CDH1 or by itself, leading to the transition from APC/C-CDC20 to APC/C-CDH1 (Nat Rev Cancer (2006) 6(5):369-381). 3. Cyclin B, the primary mitotic cyclin in multicellular animals (metazoans), is synthesized throughout the cell cycle from a stable mRNA. The observed fall in its concentration in late anaphase results from its APC-stimulated degradation at this point in the cell cycle. Genetic studies with yeast led to identification of an APC specificity factor, called Cdh1, that binds to APC and directs it to polyubiquitinate mitotic cyclins. This specificity factor is active only in late anaphase when the segregating chromosomes have moved far enough apart in the dividing cell to assure that both daughter cells will contain one complete set of chromosomes. Phosphorylation of Cdh1 by other cyclin-CDK complexes during G1 inhibits its association with the APC and thus degradation of mitotic cyclin (Figure 19-30). This inhibition permits the gradual rise in mitotic cyclin levels observed throughout interphase of the next cell cycle.

Figure 19.16 Regulation of S phase and mitotic cyclin levels in budding yeast.

Regulation of S phase and mitotic cyclin levels in budding yeast One of the important substrates of the G1/S phase cyclin-CDK complexes is Cdh1. During late anaphase, this substrate targeting factor directs the APC/C to ubiquitinylate substrate protein including S phase and mitotic cyclins, marking them for proteolysis by proteasomes. The APC/C-Cdh1 remains active throughout G1, preventing the premature accumulation of S phase and mitotic cyclins. 1. Phosphorylation of Cdh1 by G1/S cyclin-CDKs causes it to dissociate from the APC/C complex,

inhibiting further ubiquitinylation of S phase and mitotic cyclins during late G1. This, combined with the induced transcription of S phase cyclins during late G1, allows S phase cyclin proteins to accumulate as G1/S cyclin-CDKs levels rise. Later in the cell cycle, S phase and mitotic CDKs take over to maintain Cdh1 in the phosphorylated and hence inactive state.

2. Only as mitotic CDKs decline and a protein phosphatase known as Cdc14 is activated are these inhibitory phosphates removed from Cdh1, leading to its reactivation. In mammalian cells similar mechanisms are responsible for stabilizing S phase and mitotic cyclins, but the phosphatase involved in dephosphorylation of Cdh1 has not been identified (Figure 19-16).

Two experiments that demonstrate the requirement for protein degradation to exit from mitosis 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. 1. An APC inhibitor was added to frog egg extracts undergoing mitosis in vitro. The inhibitor arrested

mitosis at metaphase, indicating that proteolysis is required for the separation of sister chromatids at the metaphase-to-anaphase transition. A similar arrest occurs in budding yeasts with mutations in components of the APC.

2. A nondegradable mutant form of M-cyclin was added to mitotic frog egg extracts. This addition arrested mitosis after sister-chromatid separation, indicating that destruction of M-cyclin is not required for sister-chromatid separation but is required for the subsequent exit from mitosis.

(Figure 17-25 Molecular Biology of the Cell - http://www.ncbi.nlm.nih.gov/books/NBK26856/#A3223)

Figure 17-20b Molecular Biology of the Cell (© Garland Science 2008)

The cell-cycle control system depends on cyclical proteolysis 1. Cell-cycle control depends crucially on at least two distinct enzyme complexes that act at different times in the cycle to cause the proteolysis of key proteins of the cell-cycle control system, thereby inactivating them. Most notably, cyclin-Cdk complexes are inactivated by regulated proteolysis of cyclins at certain cell-cycle stages. This cyclin destruction occurs by a ubiquitin-dependent mechanism, like that involved in the proteolysis of many other intracellular proteins. An activated enzyme complex recognizes specific amino-acid sequences on the cyclin and attaches multiple copies of ubiquitin to it, marking the protein for complete destruction in proteasomes. 2. The rate-limiting step in cyclin destruction is the final ubiquitin-transfer reaction catalyzed by enzymes known as ubiquitin ligases. Two ubiquitin ligases are important in the destruction of cyclins and other cell-cycle regulators. ① In G1 and S phase, an enzyme complex called SCF (after its three main protein subunits) is

responsible for the ubiquitylation and destruction of G1/S-cyclins and certain CKI proteins that control S-phase initiation.

② In M phase, the anaphase-promoting complex (APC) is responsible for the ubiquitylation and proteolysis of M-cyclins and other regulators of mitosis.

3. These two large, multisubunit complexes contain some related components, but they are regulated in different ways. SCF activity is constant during the cell cycle. Ubiquitylation by SCF is controlled by changes in the phosphorylation state of its target proteins: only specifically phosphorylated proteins are recognized, ubiquitylated, and destroyed. APC activity, by contrast, changes at different stages of the cell cycle. APC is turned on mainly by the addition of activating subunits to the complex. (Figure 17-20b Molecular Biology of the Cell - http://www.ncbi.nlm.nih.gov/books/NBK26824/#A3210)


G1 CDK

G1 CDK 1. When cells are stimulated to replicate, G1 cyclin-CDK complexes are expressed first. These prepare the cell for the S phase by activating transcription factors that promote transcription of genes encoding enzymes required for DNA synthesis and the genes encoding S-phase cyclins and CDKs. During early G1 of the next cell cycle, phosphatases dephosphorylate the proteins that form pre-replication complexes. These proteins had been phosphorylated by S-phase cyclin-CDK complexes during the previous S phase, and their phosphorylation was maintained during mitosis by mitotic cyclin-CDK complexes. As a result of their dephosphorylation in G1, new pre-replication complexes are able to reassemble at replication origins in preparation for the next S phase (Figure 19-30). Phosphorylation of Cdh1 by G1 cyclin-CDK complexes in late G1 inactivates it, allowing accumulation of S-phase and mitotic cyclins during the ensuing cycle. The activity of S-phase cyclin-CDK complexes is initially held in check by inhibitors. Late in G1, the G1 cyclin-CDK complexes induce degradation of the S-phase inhibitors by phosphorylating them and consequently stimulating their polyubiquitination by the multiprotein SCF ubiquitin ligase. Subsequent degradation of the polyubiquitinated S-phase inhibitor by proteasomes releases active S-phase cyclin-CDK complexes. 2. Cyclin D and E are the mammalian G1 cyclins. Experiments in which cultured mammalian cells were microinjected with anti-cyclin D antibody at various times after addition of growth factors demonstrated that cyclin D is essential for passage through the restriction point. Figure 20-32 (6th ed.) presents a current model for the periods of the cell cycle in which different cyclin-CDK complexes act in G0-arrested mammalian cells stimulated to divide by the addition of growth factors. In the absence of growth factors, cultured G0 cells express neither cyclins nor CDKs; the absence of these critical proteins explains why G0 cells do not progress through the cell cycle and replicate.

Figure 19.15 Control of the G1–S phase transition

Control of the G1–S phase transition 1. In yeast, the G1 cyclin gene is called CLN3 (Figure 19-15a). Its mRNA is produced at a nearly constant level throughout the cell cycle, but its translation is regulated in response to nutrient levels and, as we will see shortly, CLN3 is a linchpin in coupling cell cycle entry to nutrient signals. Once sufficient Cln3 is synthesized from its mRNA, Cln3-CDK complexes phosphorylate and inactivate the transcriptional repressor Whi5. Phosphorylation of Whi5 promotes its export out of the nucleus, allowing the transcription factor complex SBF to induce transcription of the G1/S phase cyclin genes CLN1 and CLN2 as well as other gene important for DNA replication. Once produced Cln1/2-CDKs contribute to further Whi5 phosphorylation. This positive feedback loop ensures the rapid accumulation of G1/S phase CDKs. Once a critical level of Cln1/2-CDKs is reached, these G1/S phase CDKs promote bud formation, entry into S phase, and the duplication of the centroseom (also known as the spindle pole body, which later in the cell cycle will organize the mitotic spindle). This state of G1/S phase CDK activity, sufficient to initiate S phase, bud formation, and centrosome duplication, is the molecular definition of START. 2. Some members of a small family of related transcription factors, referred to collectively as E2F factors, are encoded by delayed-response genes. These transcription factors activate genes encoding many of the proteins involved in DNA and deoxyribonucleotide synthesis. They also stimulate transcription of genes encoding the late-G1 cyclin (cyclin E), the S-phase cyclin (cyclin A), and the S-phase CDK (CDK2). Thus the E2Fs function in late G1 similarly to the S. cerevisiae transcription factors SBF and MBF. In addition, E2Fs auto stimulate transcription of their own genes. E2Fs function as transcriptional repressors when bound to Rb protein, which in turn binds histone deacetylase complexes. Transcription of a gene is highest when the associated histones are highly acetylated; histone deacetylation causes chromatin to assume a more condensed, transcriptionally inactive form. Rb protein is one of the most significant substrates of mammalian G1 cyclin-CDK complexes. Phosphorylation of Rb protein at multiple sites prevents its association with E2Fs, thereby permitting E2Fs to activate transcription of genes required for entry into S phase. As shown in Figure 19-15b, phosphorylation of Rb protein is initiated by cyclin D-CDK4 and cyclin D-CDK6 in mid G1.

Regulation of Rb and E2F activities 1. Mitogen withdrawal prior to passage through the restriction point leads to accumulation of p16. p16 binds specifically to CDK4 and CDK6, thereby inhibiting their kinase activity and causing G1 arrest. Under normal circumstances, phosphorylation of Rb protein is initiated midway through G1 by active cyclin D-CDK4 and cyclin D-CDK6 complexes. Rb phosphorylation is completed by other cyclin-CDK complexes in late G1, allowing activation of E2F transcription factors, which stimulate transcription of genes encoding proteins required for DNA synthesis. The complete phosphorylation of Rb irreversibly commits the cell to DNA synthesis. Most tumors contain an oncogenic mutation that causes overproduction or loss of one of the components of this pathway such that the cells are propelled into the S phase in the absence of the proper extracellular growth signals. 2. Once cyclin E and CDK2 are induced by phosphorylation of some Rb, the resulting cyclin E-CDK2 further phosphorylates Rb in late G1. When cyclin E-CDK2 accumulates to a critical threshold level, further phosphorylation of Rb by cyclin E-CDK2 continues even when cyclin D-CDK4/6 activity is removed. This is one of the principle biochemical events responsible for passage through the restriction point. At this point, further phosphorylation of Rb by cyclin E-CDK2 occurs even when mitogens are withdrawn and cyclin D and CDK4/6 levels fall. Since E2F stimulates its own expression and that of cyclin E and CDK2, positive cross-regulation of E2F and cyclin E-CDK2 produces a rapid rise of both activities in late G1. As they accumulate, S-phase cyclin-CDK and mitotic cyclin-CDK complexes maintain Rb protein in the phosphorylated state throughout the S, G2, and early M phases. After cells complete anaphase and enter early G1 or G0, the fall in cyclin-CDK levels leads to dephosphorylation of Rb by unopposed phosphatases. As a consequence, hypophosphorylated Rb is available to inhibit E2F activity during early G1 of the next cycle and in G0-arrested cells (Figure 25-25 6th ed).

Mechanisms controlling S-phase initiation in animal cells 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. This transcriptional control system, like so many other control systems that regulate the cell cycle, includes feedback loops that sharpen the G1/S transition : The liberated E2F increases the transcription of its own gene. E2F-dependent transcription of G1/S-cyclin and S-cyclin genes leads to increased G1/S-Cdk and S-Cdk activities, which in turn increase Rb phosphorylation and promote further E2F release, forming a positive feedback loop. The increase in G1/S-Cdk and S-Cdk activities enhances the phosphorylation of Hct1 and p27, leading to their inactivation or destruction. The resulting appearance of G1/S-Cdk and S-Cdk activities further enhances Rb phosphorylationE2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop (Figure 17-62 (part 3 of 3) Molecular Biology of the Cell - http://www.ncbi.nlm.nih.gov/books/NBK26856/#A3232)

Figure 19.17 Control of S phase onset in S. cerevisiae by regulated proteolysis of the S phase inhibitor, Sic1.

Figure 19.18 Six suboptimal G1/S phase CDK phosphorylation sites in Sic1 create a switch-like cell cycle entry.

S phase inhibitor, Sic1 1. We can now see that regulated proteasomal degradation directed by two ubiquitin ligase complexes, SCF and APC, controls three major transitions in the cell cycle: onset of the S phase through degradation of Sic1, the beginning of anaphase through degradation of securin, and exit from mitosis through degradation of B-type cyclins. The APC is directed to polyubiquitinate securin, which functions as an anaphase inhibitor, by the Cdc20 specificity factor. Another specificity factor, Cdh1, targets APC to B-type cyclins. The SCF is directed to polyubiquitinate Sic1 by a different mechanism, namely, phosphorylation of Sic1 by a G1 cyclin-CDK (Figure 19-17, 19-30). This difference in strategy probably occurs because the APC has several substrates, including securin and B-type cyclins, which must be degraded at different times in the cycle. In contrast, entry into the S phase requires the degradation of only a single protein, the Sic1 inhibitor (Table 19-2). An obvious advantage of proteolysis for controlling passage through these critical points in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed irreversibly in one direction through the cycle. 2. Degradation of Sic1 is induced by its phosphorylation by G1/S phase CDKs (Figure 19-17). It must be phosphorylated at least six sites, which are relatively poor substrates for the G1/S phase CDKs, before it is bound sufficiently well by SCF to be ubiquitinylated. Multiple, poor G1/S phase CDK phosphorylation sites lead to an ultrasensitive, switch-like response in Sic1 degradation and hence precipitous activation of S phase CDKs (Figure 19-18). If Sic1 were inactivated following the phosphorylation of a single site, Sic1 molecules would start to get phosphorylated as soon as the levels of G1/S phase CDK activity begin to rise, leading to a gradual decrease in Sic1 levels. In contrast, when several sites need to be phosphorylated, at low levels of G1/S phase CDK activity only a few sites are phosphorylated and Sic1 is not destroyed. Only when G1/S phase CDK levels are high is Sic1 sufficiently phosphorylated at multiple sites to target it for degradation. Thus Sic1 degradation occurs only when G1/S phase CDK activity has reached its peak and virtually all other G1/S phase CDK substrates have been phophorylated.

Figure 19.19 The molecular mechanisms governing the initiation of DNA replication.

The molecular mechanisms governing the initiation of DNA replication 1. Eukaryotic chromosomes are replicated from multiple origins. Yeast replication origins contain an 11-bp conserved core sequence to which is bound a hexameric protein, the originrecognition complex (ORC), required for initiation of DNA synthesis. Cdc6 and Cdt1 are required to load opposing MCM helicase complex on the origin (Figure 19-19, step 1). The MCM helicase function to unwind the DNA during the initiation of DNA replication. To ensure that origins fire only once at the beginning of S phase, MCM helicase complex loading and its activation occur at two opposing phosphorylation states. ① The MCM helicases can only load onto the DNA in a state of low CDK activity that occurs when CDKs

are inactivated during exit from mitosis and during early G1. In other words, MCM helicases load onto DNA when they are unphosphorylated.

② In contrast, activation of MCM helicases and the recruitment of DNA polymerases to the unwound origin DNA are triggered by S phase CDKs. Recall that S phase CDKs only become active when G1/S phase CDK levels reach their peak and CKIs of S phase CDKs and a second heterodimeric protein kinase, DDK, activates the MCM helicase and recruits the polymerases to the sites of replication initiation (Figure 19-19, steps 2 and 3).

2. So how do S phase CDKs and DDK collaborate to initiate DNA replication? ① ORC and the two other initiation factors, Cdc6 and Cdt1, recruit the MCM helicases to sites of

replication initiation during G1, when CDK activity is low (Figure 19-19, step 1). ② When DDK and S phase CDKs are activated in late G1, DDK phosphorylates two subunits of the MCM

helicase. The S phase CDKs phosphorylate two proteins called Sld2 and Sld3. These phosphorylation events have an activating effect, promoting the recruitment of MCM helicase activators to sites of replication initiation (the phosphorylation events shown in green in Figure 19-19, steps 2 and 3).

③ The helicase activators are called the Cdc45-Sld3 complex and the GINS complex. Exactly how they promote activation of the MCM helicases is not yet clear. In addition to activating the MCM helicase to unwind DNA, the Cdc45-Sld3 complex and the GINS complex recruit polymerases to the DNA polymerase ε to synthesize the leading strand and polymerase δ to synthesize the lagging strand (see Figure 19-19, step 3). The replication machinery then initiates DNA synthesis.

Figure 19.20 Model for establishing cohesin linkage of sister chromatids.

Model for establishing cohesin linkage of sister chromatids The linkages between sister chromatids established during S phase will be essential for their accurate segregation during mitosis. The protein complexes that establish cohesion between sister chromatids are called cohesins. Some details of how cohesins are loaded onto DNA to mediate sister chromatid cohesion are being unraveled. We know that establishment of cohesion between sister chromatids is tightly tied to DNA replication. Cohesins associate with chromosomes during G1 (Figure 19-20, step 1). During DNA replication, they are loaded onto chromosomes in such a way that they can hold sister chromatids together. Most likely this occurs as replication forks replicate the DNA (step 2). Converting DNA-bound G1 cohesins into cohesive complexes requires several cohesin loading factors-including a protein complex related to proteins that load the sliding clamp at the replication fork-and the acetylation of the Smc3 subunit.

1. Step1: Cohesins are loaded onto chromosomes during G1, but they do not posses cohesive properties

(indicated as cohesins laterally associated with chromosomes). 2. Step2: Concomitant with DNA replication, and most likely closely behind the replication fork, cohesins

are converted into cohesive molecules, able to hold sister chromatids together (indicated as cohesin rings encircling the replicated sister chromatids). This conversion into cohesive cohesins requires cohesin loading factors. During G2, sister chromatids are replicated and linked along their entire length by cohesins. During this time, the Mei-S332/Sgo proteins recruit the protein phosphatase 2A (PP2A) to centromeric regions.

3. Step3: In vertebrate cells, cohesins are released from chromosome arms during prophase and early metaphase by the action of Polo kinase and Aurora B kinase. By the end of metaphase, cohesins are retained only in the region of the centromere, where PP2A prevents cohesin phosphorylation and hence dissociation through recruiting PP2A.

Figure 19.21 Phosphorylation on the CDK subunit restrains mitotic CDK activity during S phase and G2.

Phosphorylation on the CDK subunit restrains mitotic CDK activity during S phase and G2 Mitotic CDKs initiate mitosis. Whereas levels of the catalytic CDK subunit are constant throughout the cell cycle, mitotic cyclins gradually accumulate during S phase. Most eukaryotes contain multiple mitotic cyclins, which for historical reasons are called cyclin A and cyclin B. As they assemble, mitotic CDK complexes are maintained in an inactive state through inhibitory phosphorylation on the CDK subunit. Two highly conserved tyrosine and threonine residues in mammalian CDKs are subject to regulated phosphorylation. In CDK1, the mitotic CDK, phosphorylation of tyrosine 15 and threonine 14 maintains mitotic cyclin-CDK complexes in an inactivate state. The phosphorylation state of T14 and T15 is controlled by a dual-specificity protein kinase known as Wee1 and a dual-specificity phosphatase Cdc25 (Figure 19-21). The regulation of mitotic CDKs by these activities underlies the abrupt activation of their kinase activity at the G2-M phase transition and explains the observation that although mitotic cyclins gradually accumulate during S phase and G2, mitotic CDKs are not active until cells enter mitosis. Activation of mitotic CDKs is the consequence of rapid inactivation of Wee1 and activation of Cdc25. Central to this rapid transition are feedback loops, where mitotic CDKs activate Cdc25 and inactivate Wee1 (Figure 19-21). Phosphorylation of Cdc25 by mitotic CDKs stimulates its phosphatase activity; phosphorylation of Wee1 by mitotic CDKs inhibits its kinase activity. Ongoing DNA replication inhibits Cdc25 activity. A critical question that we know little about is how this positive feedback loop is started once DNA replication has been completed. CDKs that function earlier in the cell cycle have been suggested to start the positive feedback loop.

Figure 19.34 Overview of DNA damage checkpoint controls in the cell cycle.

DNA damage checkpoint controls in the cell cycle 1. The complete and accurate duplication of the genetic material is essential for cell division. If cells enter mitosis when DNA is not completely replicated or otherwise damaged, genetic changes occur. In many instances, those changes will lead to cell death, they can also lead to genetic alterations that result in loss of growth and division control and eventually cancer. The enzymes that replicate DNA are highly accurate, but this exactness is not enough to ensure complete accuracy during DNA synthesis. Furthermore, environmental insults such as x-rays and UV light can cause DNA damage, and this damage must be repaired before a cell's entry into mitosis. Cells have a DNA damage response system in place that senses many different types of DNA damage and responds by activating repair pathways and halting cell cycle progression until the damage has been repaired. Cell cycle arrest can occur either in G1, S phase, or G2, depending on whether DNA damage occurred before cell cycle entry or during DNA replication. 2. Chk1 and Chk2 halt the cell cycle. The protein kinases phosphorylate Cdc25, thereby inactivating it. When the DNA damage occurs during G1, Cdc25A inhibition results in inhibition of G1/S phase CDKs and S phase CDKs (Figure 19-34). ① As a result, these kinases cannot initiate DNA replication. When the DNA damage occurs during S phase or in G2,

Cdc25C inhibition by Chk1/2 results in the inhibtion of mitotic CDKs and hence arrest in G2. ② Active DNA replication also inhibits entry into mitosis. ATR continues to inhibit Cdc25C via Chk1 until all replication forks

complete DNA replication and disassemble. This mechanism makes the initiation of mitosis dependent on the completion of chromosome replication.

③ Finally, cells also sense DNA replication stress that results in stalled or slowing of replication forks. It triggers activation of the ATR-Chk1 checkpoint pathway and results in down-regulation of S phase CDK activity and prevents firing of late-replicating origins.

④ Chk1-mediated inhibition of the Cdc25 family of phosphatases is not the only mechanism whereby DNA damage or incomplete replication inhibits cell cycle progression. DNA damage leads to the activation of the transcription factor p53, which transcribes the CDK inhibitor p21. p21 binds to and inhibits all metazoan cyclin-CDK complexes. As a result, cells are arrested in G1 and G2 (Figure 19-34). Under some circumstances, such as when DNA damage is extensive, p53 also activates expression of genes that lead to apoptosis, the process of programmed cell death that normally occurs in specific cells during the development of multicellular animals.

Figure 19.33 The DNA damage response system.

The DNA damage response system 1. DNA damage exists in many different forms and ranges in severity. A break of the DNA helix, known as a double-strand break, is perhaps the most severe form of damage since such a lesion would almost certainly lead to DNA loss if mitosis ensued in its presence. More subtle defects include single-strand breaks, structural changes in nucleotides, or DNA mismatches. For our discussion here, it is important to note that cells have sensors for all these different types of damage. These sensors scan the genome and, when they detect a lesion, assemble signaling and repair factors onto the site of the lesion. 2. Central to the detection of the different lesions is a pair of homologous protein kinase called ATM and ATR. These protein kinases get recruited to sites of DNA damage. They then initiate the sequential recruitment of adapter proteins and another set of protein kinases called Chk1 and Chk2. These kinases then activate repair mechanisms and cause cell cycle arrest or apoptosis in animals (Figure 19-33). ATR and ATM recognize different types of DNA damage. ATM is very specialized in that it only responds to double-strand breaks. ATR is able to recognize more diverse types of DNA damage, such as stalled replication forks, damaged nucleotides, and double-strand breaks. Stalled replication forks, for example, are recognized by ATR. The association of ATR with stalled forks is thought to activate its protein kinase activity, leading to the recruitment of adapter proteins whose function is to recruit and help activate the Chk1 kinase. Active Chk1 then induces repair pathways and inhibits cell cycle progression. 3. A key effector of the DNA damage response in metazoan cells is the transcription factor p53 (Figure 19-33). It is known as a tumor suppressor because its normal function is to limit cell proliferation in the face of DNA damage. The protein is extremely unstable and generally does not accumulate to high enough levels to stimulate transcription under normal conditions. The instability of p53 results from its unbiquitinylation by a ubiquitin-protein ligase called Mdm2 and subsequent proteasomal degradation. The rapid degradation of p53 is inhibited by ATM and ATR, which phophorylate p53 at a site that interferes with Mdm2 binding. This and other modifications of p53 in response to DNA damage greatly increase its ability to activate transcription of specific genes that help the cell cope with DNA damage. One of these genes encodes the CKI p21.

How DNA damage arrests the cell cycle in G1 1. 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. 2. 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 17-63 Molecular Biology of the Cell - http://www.ncbi.nlm.nih.gov/books/NBK26856/)

Nat Rev Cancer (2007) 7(11):819-822

A model of the p53–miR- 34 network in regulating cell proliferation and cell death 1. miRNAs act by inhibiting gene expression. First identified as small, non-coding RNAs essential for the timing of larval development in worms, a large number of miRNAs have been and are still being discovered in nearly all metazoans. Most animal miRNAs share common biogenesis and effector machineries. Mature miRNAs often range from 20–22 nucleotides in length as a result of two sequential processing reactions, which liberate mature species from their stem-loop precursors. After maturation, these small RNAs are incorporated into the RNA-induced silencing complex (RISC), through which they mediate post-transcriptional gene silencing of specific mRNA targets. 2. Recent studies have identified miR-34 as a microRNA (miRNA) component of the p53 network, for the first time revealing an interplay between proteins and non-coding RNAs in this crucial tumour-suppressor pathway. miR-34 is a direct transcriptional target of p53, which in turn downregulates genes required for proliferation and survival. Along with other p53 targets, such as p21 and BAX, miR-34-family miRNAs promote growth arrest and cell death in response to cancer related stress (Nat Rev Cancer (2007) 7(11):819-822).

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