In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signaling and DSB repair by homologous recombination (HR). Previous work has implicated the MRE11 complex in such DSB processing events. Here, we show that the human CtIP protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in S/G2. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of RPA and ATR to DSBs and ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient HR. Finally, we reveal that CtIP displays sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signaling and HR.DSBs are highly cytotoxic lesions induced by ionizing radiation and certain anti-cancer drugs. They also arise when replication forks encounter other lesions and are generated as intermediates during meiotic recombination1. Cells possess two main DSB repair mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). While NHEJ predominates in G0/G1 and is error-prone, HR is restricted to S and G2, when sister chromatids allow faithful repair2-4. HR is initiated by resection of DSBs to generate single-stranded DNA (ssDNA) that binds RPA. A ssDNA-RAD51 nucleoprotein filament then forms to initiate strand invasion. ssDNA-RPA also recruits the protein kinase ATR, triggering ATR-dependent checkpoint signaling by the protein kinase Chk15. As DSB resection is largely restricted to S and G2, both HR and checkpoint signaling are subject to cell-cycle control6-8.A factor implicated in DSB resection is the MRE11-RAD50-NBS1 (MRN) complex, which binds DNA ends, possesses exo-and endo-nuclease activities and functions in triggeringCorrespondence and requests for materials should be addressed to: Stephen P. Jackson 1 , Email: s.jackson@gurdon.cam.ac.uk, Telephone: +44 (0)1223 334088, Fax: +44 (0)1223 334089. Author contributions S.F and R.B generated CtIP cDNA, CtIP antibodies and recombinant CtIP protein. C.L., J.L., M.M and J.B generated the cell lines with GFP-tagged proteins, conceived, performed and evaluated the real-time imaging experiments, and performed the HR measurements. All other experiments were conceived by A.A.S and S.P.J, and were performed by A.A.S with the help of J.C. A.A.S and S.P.J wrote the paper. All authors discussed the results and commented on the manuscript. Author informationThe authors declare no competing financial interest. Europe PMC Funders Group CtIP affects cellular responses to DSBsTo investigate CtIP function, we examined how its depletion affected clonogenic survival of human U2OS cells following their treatment with DNA-damaging agents. To circumvent possible effects arising from CtIP's involveme...

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family, and are rapidly activated in response to DNA damage. ATM and DNA-PKcs respond mainly to DNA double-strand breaks, whereas ATR is activated by single-stranded DNA and stalled DNA replication forks. In all cases, activation involves their recruitment to the sites of damage. Here we identify related, conserved carboxy-terminal motifs in human Nbs1, ATRIP and Ku80 proteins that are required for their interaction with ATM, ATR and DNA-PKcs, respectively. These motifs are essential not only for efficient recruitment of ATM, ATR and DNA-PKcs to sites of damage, but are also critical for ATM-, ATR- and DNA-PKcs-mediated signalling events that trigger cell cycle checkpoints and DNA repair. Our findings reveal that recruitment of these PIKKs to DNA lesions occurs by common mechanisms through an evolutionarily conserved motif, and provide direct evidence that PIKK recruitment is required for PIKK-dependent DNA-damage signalling.