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Chapter 21 : Cell Cycle Checkpoints
Category: Microbial Genetics and Molecular Biology
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This chapter illustrates the strategies of signal amplification and transmission and the multiple targets in cell cycle and repair in various species. The signal transduction mechanisms rely on the activation of protein kinases as effector proteins by phosphorylation. Other proteins, termed mediators, are critical for converting sensor input into such kinase modification. The best-understood example for such collaboration is represented by the Rad53 kinase and Rad9 as its mediator of checkpoint activation in Saccharomyces cerevisiae. It discusses the downstream targets that are regulated by the damage-sensing and signal transmission systems. The discussion of targets involved in cell cycle progression is separated from the discussion of targets affecting aspects of repair more directly. The chapter also discusses p53 in damage-signaling pathways, and presents an integrated view of various cell cycle checkpoints and their regulation. In conclusion, it is appropriate to mention at least two additional areas, which arguably can be classified as checkpoint responses; these are the active inhibition of transcription and translation in response to DNA damage.
Domain structure of Rad53/Cds1/CHK2 kinases. Locations of FHA, kinase, and SQ/TQ-rich domains in the various orthologs are shown. The star indicates the highly conserved activation loop in each ortholog. aa, amino acids. (Adapted from reference 24 .)
Role of S. cerevisiae Rad9 as a mediator of Rad53Sc kinase activation. In this model, activation of Mec1Sc (targeted by Lcd1Sc) has already occurred and substrate selection by interaction with damage sensor proteins has been enabled, possibly by a mechanism similar to the one shown in Fig. 20–15. Mec1Sc phosphorylates Rad9Sc, which binds near damaged DNA sites and forms dimers or multimers. Phosphorylated Rad9Sc is bound by Rad53Sc. Rad53Sc will autophosphorylate in trans, possibly after initial direct phosphorylation and activation by Mec1Sc. Hyperphosphorylated and fully active Rad53Sc species emerge which separate from Rad9Sc, and Rad9Sc is free to interact again with hypophosphorylated Rad53Sc.
Domain structure of S. cerevisiae Rad9. The locations of the tandem BRCT repeat, potential nuclear localization signals (NLS), the Rad53Sc-interacting region, and an SC cluster domain (SCD) are shown. Additionally, potential phosphorylation sites for the budding-yeast Cdk Cdc28 and for PI3 kinase (PI3K)-like kinases such as Mec1Sc are indicated. (Adapted from reference 406 .)
Similarities of the roles of mammalian MDC1 and 53BP1. Both are phosphorylated by ATM, both are suspected to be possible mediators of ATM-dependent CHK2 activation, and both interact physically and functionally with -γ-H2AX. CHK2 activation by autophosphorylation is not shown here (see Fig. 21–3 ). Phosphorylation of H2AX depends on ATM following DSB induction and on ATR and its targeting factor ATRIP following UV radiation. MDC1 interacts with the MRN complex and strengthens ATM binding to DSB.
Checkpoint kinase-mediator relationships in selected eukaryotic organisms. ?, unknown or unproven candidate. (Adapted from reference 77 .)
UV irradiation enhances the half-life of p53 protein. Proteins were labeled with [35S] methionine in mouse cells before treatment of the cells with UV radiation at 0 (—UV) or 10 (+UV) J/m2. Relative steady-state levels of p53 were detected by immunoprecipitation at various time points during postirradiation incubation in the presence of nonradioactive methionine. These chase periods are indicated on the x axis. (Adapted from reference 261 .)
Selected post-translational modifications of p53 in relation to domain structure and protein interactions in response to various stresses that may or may not be associated with geno-toxicity. The protein’s transactivation, SH3 (SRC homology 3-like), DNA-binding, tetramerization (Tet), regulatory domains (Reg), nuclear localization signal (NLS), and nuclear export signal (NES) are shown. Regions of protein interaction are indicated below the map. Post-translational modification sites are indicated for phosphorylation (P), acetylation (Ac), methylation (Me), ubiquitination/NEDDylation (Ub, Ub/N), and sumoylation (SUMO). Putative modifying enzymes are listed (? indicates an unknown enzyme). In the upper half, the available studies of the influence of the treatments listed to the left on the modification of each site are summarized. An open symbol stands for no detectable modification, and a solid symbol stands for an increase in modification under the condition listed. A down arrow in a symbol stands for downregulation. A solid symbol with asterisks indicates ATM-dependent phosphorylation. (Adapted from reference 11a and C. W. Anderson, personal communication.)
IR-induced p53 phosphorylation at certain sites is reduced in AT cells. Immunoblots with the indicated phosphospecific polyclonal antibodies were performed at the times indicated following 8-Gy γ-irradiation (IR) treatment of human lymphoblast cultures. Reduced phosphorylation was found for Ser9, Ser15, Ser20, and Ser46 in AT cells but not or much less so for Ser6, Ser33, Ser315, and Ser392. The first row shows the level of total p53 detected with a monoclonal antibody. (Adapted from reference 335 .)
Defective G1 arrest in p53-deficient cells following exposure to IR. (A) The dot diagram visualizes the distribution of cells among different cycle stages in an unsynchronized culture of embryonic mouse fibroblasts before and after X-ray treatment. Cells were pulse-labeled with BrdU and analyzed by flow cytometry (see Fig. 20–8). The y axis indicates total DNA content (detected by propidium iodide staining), whereas the x axis shows the amount of incorporated BrdU (detected by binding of a BrdU-specific antibody conjugated to a fluorescent dye). Cells in S phase are characterized by high BrdU levels and an intermediate total DNA content. A depletion of cells in S phase is evident 16 h after irradiation with 2 Gy of normal cells (wt/wt) and those carrying a heterozygous p53 defect (wt/ —). However, there is no indication of G1/S arrest in cells homozygous for a p53 defect (— / —). (B) This type of analysis is quantitated by plotting the percentage of cells remaining in S phase 16 h after irradiation with 0 or 2 Gy. (Adapted from reference 189 .)
The kinase activity of a Cdk2-cyclin A complex is inhibited by increasing amounts of p21. Kinase activity is measured by histone H1 phosphorylation with 32P (H1-p). (Adapted from reference 146 .)
Scheme for p53-mediated maintenance of G1/S arrest in response to DNA damage. As described, DNA damage activates the PI3 kinase-related ATM and ATR kinases, which in turn activate CHK1 and CHK2. Modification of p53, assisted by MDM2 phosphorylation, results in p53 activation and stabilization of its level. The transcription of the p21 gene is increased. Accumulated p21 blocks CDK2-cyclin E (Cyc E) and other Cdk activities. As a consequence, RB1 is not hyperphosphorylated and the E2F transcription factor controlling S-phase-specific transcripts is not released. (Adapted from reference 26 .)
Scheme for rapid G1 arrest mechanisms in response to DNA damage. Following activation of effector kinases CHK1 and CHK2, CDC25A phosphatase is phosphorylated (left). Phosphorylation results in recognition and ubiquitylation of CDC25A by the SCF complex and ultimately to degradation by the proteasome. The absence of phosphatase CDC25 leads to a failure to dephosphorylate and activate CDK2. One consequence is the inability of CDC45 to bind to the origin (ORI) recognition complex, a necessary precondition for replicon initiation. The cell will not enter S phase from G1 or, if already in S phase, will not continue origin activation. The other proposed mechanism (right) is based on damage-induced destabilization of the cyclin D1 (CycD1) subunit of CDK4 by an unknown mechanism. In many mammalian somatic cells, abundant cyclin D1 is used to bind p21 at a stage where the dependency of cell cycle progression switches from the CDK4-cyclin D1 to the CDK2-cyclin E (Cyc E) complex (see Fig. 20–1). Destabilization of cyclin D1 results in release of p21 from this reservoir, and the CDK2-cyclin E complex will be inactivated. (Ub, ubiquitin) (Adapted from reference 26 .)
Influence of Rad53-dependent checkpoint mechanisms on S. cerevisiae DNA replication in the presence of MMS damage. G1-synchronized cells with density-labeled DNA are released into MMS-containing medium. Progression of DNA synthesis initiated at an early replicon (ARS607) of chromosome X is followed by separation of chromosomal DNA into heavy (HH) and intermediate-density (HL, heavy-light) fractions and the use of probes 1 to 6 to detect the presence of sequences downstream of the ARS607 origin. In the wild type (WT), a slow but ultimately complete shift from HH to HL density is found throughout the region, indicating complete replication of the 70-kb monitored area. Two differences are found in the checkpoint-deficient rad53 mutants (as well as in mec1 mutants [not shown]). At 60 min, there is evidence of a progressing replication fork entering distal from the studied region (from the right of area 6 [arrow 1]). The corresponding origin is silenced in the MMS-treated wild type. Also, there is evidence of a fraction of DNA that is never completely replicated (clearly visible if area 3 at 90 through 240 min is compared to the wild type [arrow 2]). This is indicative of replication fork collapse. (Adapted from reference 404 .)
Parallel pathways regulate the mammalian IR-induced S-phase checkpoint(s). One well-understood pathway functions by degradation of the CDC25 phosphatase following ATM-mediated phosphorylation. The MRN complex, BRCA1, and FANCD2 seem to be involved in alternative pathways. As discussed in the text, an important target is SMC1, which is phosphorylated by ATM in a reaction that depends on the MRN complex and BRCA1. The mechanism of SMC1 and FANCD2 action is unknown.
Regulation of S. pombe G2/M arrest in response to DNA damage. Dephosphorylation of Tyr15 (and Thr14 [not shown]) is required for activity of the mitotic Cdk Cdc2 of S. pombe. Wee1 and Mik1 kinases phosphorylate Tyr15, and Cdc25 is the activating phosphatase. Cdc25 appears to be the main target of DNA damage regulation. It is phosphorylated by the S. pombe Mec1Sc/ATR homolog Rad3Sp. Phosphorylated Cdc25 is subject to binding by the 14-3-3 protein Rad24Sp and is actively excluded from the nucleus.
Regulation of mammalian G2/M arrest in response to DNA damage. Part of this scheme (right half) depicts the regulation of CDC25 phosphatase, as shown in more detail for the S. pombe paradigm in Fig. 21–15 . To the left, the role of p53-mediated transcription in arrest maintenance is indicated. Enhanced transcription of the 14-3-3s and p21 genes results in increased inactivation and cytoplasmic sequestration of CDK1 (i.e., CDC2). (Adapted from reference 68 .)
Branched pathway of M-phase arrest in S. cerevisiae in response to DNA damage. In response to DNA strand breakage and the resulting Mec1Sc activation, branches mediated by the effector kinases Chk1Sc and Rad53Sc represent different mechanisms and timing of arrest. The Chk1Sc pathway blocks cohesin cleavage and thus chromosome separation at the metaphase-anaphase transition. The Rad53Sc pathway inhibits mitotic exit by preventing B-type cyclin degradation and other associated events. In both cases, substrate recognition and protein degradation by the anaphase-promoting complex (APC) are impaired.