Abstract
The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity.