FIGURE 14.9 Models for the mechanism of action of two DNA-damage checkpoints. AT
ID: 102027 • Letter: F
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FIGURE 14.9 Models for the mechanism of action of two DNA-damage checkpoints. ATM and ATR are protein kinases that become activated following specific types of DNA damage. Each of these proteins acts through checkpoint signaling pathways that lead to cell cycle arrest. ATM becomes activated in response to double-strand breaks, which are detected by the MRN proteir complex (step a). ATR, on the other hand, becomes activated by protein-coated SsDNA (step 1) that forms when replication forks become stalled or the DNA is being repaired after various types of damage. In the G2 pathway shown here ATR phosphorylates and activates the checkpoint kinase Chk1 (step 2), which phosphorylates and inactivates the phosphatase Cdc25 (step 3), which normally shuttles between the nucleus and cytoplasm (step 4). Once phosphorylated Cdc25 is bound by an adaptor protein in the cytoplasm (step 5) and cannot be reimported into the nucleus, which leaves the Cdk in its inactivated, phosphoryl ated state (step 6). In the G1 pathway shown here, ATM phosphorylates and activates the checkpoint kinase Chk2 (step b), which phosphorylates p53 (step c) p53 is normally very short-lived, but phosphorylation by Chk2 stabilizes the protein, enhancing its ability to activate p21 transcription (step d). Once transcribed and translated (step e), p21 directly inhibits the Cdk (step f). Many other proteins, including histone-modifying enzymes, chromatin remodeling complexes, and histone variants are involved in mediating the response to DNA damage but are not discussed SOURCE: See Curr. Opin. Cell Biol. 21:245, 2009; Nature Revs. Mol. Cell Biol. 10: 243, 2009; Nature Cell Biol. 13:1161, 2011; and Genes Develop. 25:409, 2011 Nucleus Ultraviolet radiation lonizing radiation MRN complex SSDNA-protein complex ATR ATM Inactive hk1 Chk2 Inactive Active(Chkl Chk2 Active 53 Unstable 53 Stable 6 nactive DNA P p21 gene CELL CYCLE ARREST Active p21 mRNA absence of Cdc25 from the nucleus leaves the Cdk in an inactive Inactive Cdk Cytoplasmm state (step 6) and the cell arrested in G2 Adaptor protein (1 4-3-30 CELL CYCLE ARREST 2. Damage to DNA also leads to the synthesis of proteins that directly inhibit the cyclin-Cdk complex that drives the cell cycle. For example, cells exposed to ionizing radiation in G1 synthesize nrotein called n21 (moleculms f 21 V that inhibit toExplanation / Answer
Proceeding of cell cycle with mutated DNA is deleterious to the cells. Cells may miss some important genetic material needed for their survival or further proliferation. These mutations may lead to apoptosis or even cancer. ATM and ATR are the key proteins that maintain the genomic integrity.
UV exposure to the cells, leads to the formation of adjacent thymidine dimmers in the same strand, leaving the formation of unbound single stranded DNA molecules. Recombinant proteins bind to these ssDNA molecules and immediately lead to the activation of ATR kinase. This starts the process of cell cycle arrest in G2 phase. ATR then phosphorylates and activates Chk1 kinase. Cdk needs to be phosphorylated by cdk activating kinases (CAK), in order to show its activity for crossing G2 phase. Along with activating phosphorylations Wee1 kinases also show inactivating phosphorylations. Cdc25 is a phosphatase, which removes only inactivating phosphorylations on cdk, leaving aside activating phosphorylations. This leads to complete activation of cdk/cyclin and proceeding of G2 to M transition. During DNA damage activated Chk1, phosphorylates cdc25 and facilitates its binding to 14-3-3 adapter proteins. These adapter proteins export cdc25 to cytoplasm, as a result cdk will continuously have inactivating phosphorylations and cannot proceed G2/M transition, leading to cell cycle arrest. Whenever the DNA damage is repaired, ATR cannot phosphorylate Chk1, due to which p-Chk1 will decrease and cdc25 levels will increase in the nucleus. This then dephosphorylate inactivating phosphorylations on cdk and leads to cell cycle progression.
Other than the formation of ssDNA, double strand DNA breaks is another mechanism of DNA damage, which is even more dangerous, as it leads to chromosomal rearrangements, if not repaired properly. Here MRN complex binds to DNA double strand breaks and attempts to repair. While attempting, they activate cell cycle arrest by activating ATM kinase. ATM phosphorylates another check point kinase, Chk2. p53 is a tumor suppressor gene and a transcription factor. It controls the expression of genes responsible for cell cycle arrest such as p21. This p21 directly binds to cdk and inhibits its binding to cyclins, effectively inhibiting G1 to S transition. In normal scenario, p53 will always be formed and degraded immediately in Mdm2 dependent manner. So it cannot always activate p21 and cell cycle arrest. But during DNA damage, Chk2 phosphorylates p53. This phosphorylation inhibits p53 binding to Mdm2, which then cannot degrade p53, leading to its stabilization and cell cycle arrest in p21 dependent fashion. When the DNA is repaired p-Chk2 levels will go down, p53 will be degraded, p21 will not be formed and ultimately leads to G1 to S cell cycle progression.
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