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.
