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The ATM- and Rad3-related (ATR) kinase is a master regulator of the DNA damage response, yet how ATR is activated toward different substrates is still poorly understood. Here, we show that ATR phosphorylates Chk1 and RPA32 through distinct mechanisms at replication-associated DNA double-stranded breaks (DSBs). In contrast to the rapid phosphorylation of Chk1, RPA32 is progressively phosphorylated by ATR at Ser33 during DSB resection prior to the phosphorylation of Ser4/Ser8 by DNA-PKcs. Surprisingly, despite its reliance on ATR and TopBP1, substantial RPA32 Ser33 phosphorylation occurs in a Rad17-independent but Nbs1-dependent manner in vivo and in vitro. Importantly, the role of Nbs1 in RPA32 phosphorylation can be separated from ATM activation and DSB resection, and it is dependent upon the interaction of Nbs1 with RPA. An Nbs1 mutant that is unable to bind RPA fails to support proper recovery of collapsed replication forks, suggesting that the Nbs1-mediated mode of ATR activation is important for the repair of replication-associated DSBs.
Shiotani et al. (Wed,) studied this question.