DNA damage initiates a signaling pathway by activating one of a pair of related protein kinases called ATM and ATR, which associate with the site of damage and phosphorylate various target proteins, including two other protein kinases called Chk1 and Chk2. These various kinases phosphorylate other target proteins that lead to cell-cycle arrest. A major target is the gene regulatory protein p53, which stimulates transcription of the gene encoding p21, a CKI protein; p21 binds to G1/S-Cdk and S-Cdk complexes and inhibits their activities, thereby helping to block entry into the cell cycle (Figure 17–62 and Movie 17.8).
DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations. This is largely because it interacts with another protein, Mdm2, which acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. Phosphorylation of p53 after DNA damage reduces 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 (see Figure 17–62).
The protein kinases Chk1 and Chk2 also block cell-cycle progression by phosphorylating members of the Cdc25 family of protein phosphatases, thereby inhibiting their function. As described earlier, these phosphatases are particularly important in the activation of M-Cdk at the beginning of mitosis (see Figure 17–20). Chk1 and Chk2 phosphorylate Cdc25 at inhibitory sites that are distinct from the phosphorylation sites that stimulate Cdc25 activity. The inhibition of Cdc25 activity by DNA damage helps block entry into mitosis (see Figure 17–16).
The DNA damage response can also be activated by problems that arise when a replication fork fails during DNA replication. When nucleotides are depleted, for example, replication forks stall during the elongation phase of DNA synthesis. To prevent the cell from attempting to segregate partially replicated chromosomes, the same mechanisms that respond to DNA damage detect the stalled replication forks and block entry into mitosis until the problems are resolved.
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 DNA damage response is not functioning. Over the long term, the accumulation of genetic damage in cells lacking the DNA damage response 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 20). This loss of p53 function allows the cancer cell stable, active p53
Figure 17–62 How DNA damage arrests the cell cycle in G1. When dna is damaged, various protein kinases are recruited to the site of damage and initiate a signaling pathway that causes cell-cycle arrest. The first kinase at the damage site is either aTm or aTR, depending on the type of damage. additional protein kinases, called Chk1 and Chk2, are then recruited and activated, resulting in the phosphorylation of the transcription regulatory protein p53. mdm2 normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. phosphorylation of p53 blocks its binding to mdm2; as a result, p53 accumulates to high levels and stimulates transcription of numerous genes, including the gene that encodes the CkI protein p21. The p21 binds and inactivates g1/s-Cdk and s-Cdk complexes, arresting the cell in g1. In some cases, dna damage also induces either the phosphorylation of mdm2 or a decrease in mdm2 production, which causes a further increase in p53 (not shown).