Abstract
Stalled replication forks induce p53, which is required to maintain the replication checkpoint. In contrast to the well-established mechanisms of DNA damage-activated p53, the downstream effectors and upstream regulators of p53 during replication blockade remain to be deciphered. Hydroxyurea triggered accumulation of p53 through an increase in protein stability. The requirement of p53 accumulation for the replication checkpoint was not due to p21(CIP1/WAF1) as its down-regulation with short-hairpin RNA did not affect the checkpoint. Similar to DNA damage, stalled replication triggered the activation of the MRN-ataxia telangiectasia mutated (ATM)/ATM and Rad3-related-CHK1/CHK2 axis. Down-regulation of CHK1 or CHK2, however, reduced p53 basal expression but not the hydroxyurea-dependent induction. Moreover, p53 was still stabilized in ataxia telangiectasia cells or in cells treated with caffeine, suggesting that ATM was not a critical determinant. These data also suggest that the functions of ATM, CHK1, and CHK2 in the replication checkpoint were not through the p53-p21(CIP1/WAF1) pathway. In contrast, induction of p53 by hydroxyurea was defective in cells lacking NBS1 and BLM. In this connection, the impaired replication checkpoint in several other genetic disorders has little correlation with the ability to stabilize p53. These data highlighted the different mechanisms involved in the stabilization of p53 after DNA damage and stalled replication forks.
Highlights
Checkpoints operate throughout the cell cycle to maintain genome stability
We found that the levels of p53 and p21CIP1/WAF1 attained after hydroxyurea treatment were significantly lower than those after DNA damage
To verify that p53 is induced during the replication checkpoint, cells were treated with different inhibitors of S phase and the expression of p53 was examined by immunoblotting www.aacrjournals.org
Summary
Checkpoints operate throughout the cell cycle to maintain genome stability. DNA damage checkpoints ensure that damaged DNA is neither replicated nor segregated to daughter cells until repaired. The replication checkpoint ensures that cells do not enter mitosis until DNA replication is completed. Cells that harbor defects in these pathways are prone to genome instability and neoplastic transformation. Genotoxic stress, including doublestrand breaks, stabilizes and activates p53. Several downstream targets of p53 are known to modulate cell proliferation. The expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1 is enhanced by p53, facilitating cell cycle arrest. Another target of p53 is the proapoptotic protein BAX, which promotes the release of cytochrome c from the mitochondria and activates the apoptotic
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