Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin

Abstract

The inhibitory effect of p53 on homologous recombination (HR) is exerted through sequestration of replication protein A (RPA). Release of the p53/RPA complex in response to replication stress is crucially dependent on the phosphorylation status of both proteins and is required for efficient DNA repair by HR. Phosphorylation of RPA within its RPA2 subunit by cyclin-dependent kinases (CDK) is an early event in the replication stress response. Here we investigated the role of transcriptional activation of the p53 downstream target, p21^Waf1, on RPA2 phosphorylation, the stability of the p53/RPA complex and HR in cells undergoing replication arrest induced by camptothecin (CPT). We show that in CPT-treated cells, activation of p53 and p21^Waf1 impedes RPA2 phosphorylation, while their depletion by siRNA stimulates it. The p53/RPA complex is more stable in wild-type cells than in cells depleted of p21^Waf1. We used nocodazole-synchronized cells treated with CPT at the entrance to S phase to assess rates of HR. Regardless of their p53 or p21^Waf1 status, the cells proceed through S phase at a similar rate and enter G2. While HR is low in wild-type cells and high in p53-depleted cells, only partial inhibition of HR is observed in the p21^Waf1-depleted cells. This correlates with the extent of RPA sequestration by p53. Thus, in CPT-treated cells, p53-induced transcriptional activation of p21^Waf1 regulates RPA2 phosphorylation, the stability of the p53/RPA complex and HR.