Activation of DSB Processing Requires Phosphorylation of CtIP by ATR

SummaryDNA double-strand breaks (DSBs) activate a DNA damage response (DDR) that coordinates checkpoint pathways with DNA repair. ATM and ATR kinases are activated sequentially. Homology-directed repair (HDR) is initiated by resection of DSBs to generate 3′ single-stranded DNA overhangs. How resection and HDR are activated during DDR is not known, nor are the roles of ATM and ATR in HDR. Here, we show that CtIP undergoes ATR-dependent hyperphosphorylation in response to DSBs. ATR phosphorylates an invariant threonine, T818 of Xenopus CtIP (T859 in human). Nonphosphorylatable CtIP (T818A) does not bind to chromatin or initiate resection. Our data support a model in which ATM activity is required for an early step in resection, leading to ATR activation, CtIP-T818 phosphorylation, and accumulation of CtIP on chromatin. Chromatin binding by modified CtIP precedes extensive resection and full checkpoint activation.

► ATR phosphorylates CtIP at a conserved site (T818 in Xenopus, T859 in human)
► This is required for stable CtIP chromatin binding and CtIP-dependent resection
► ATM activity is required prior to ATR for resection and leads to ATR activation
► This demonstrates a direct link between checkpoint activation and DSB repair