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...

SummaryThe appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5′ end resection, but how this is achieved remains uncertain. Here we report that Rif1−/− mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination.

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR) 1 . HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template [2][3][4][5] . By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs) 2,3,5,7 , with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cellcycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Δ null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.To initiate HR, one strand of the broken DNA duplex is resected in the 5′→3′ direction, generating single-stranded DNA (ssDNA) that can anneal with a homologous DNA duplex 8 . In S. cerevisiae, effective resection and HR require sustained Cdc28/Clb (Cdk1/cyclin B) kinase activity 2,3,5 , although the CDK targets mediating this control are still unknown. One potential target is Sae2, a protein first identified as being required for meiotic recombination. Sae2 controls the initiation of DNA-end resection in meiotic and mitotic cells [9][10][11][12] and was recently shown to be a DNA endonuclease 13 Fig. 2b and Supplementary Fig. 2a). In accord with this idea, the amount of slower-migrating Sae2 was diminished when Cdc28 was inactivated in G2-synchronized cultures by galactose-driven expression of the Cdc28/Clb repressor, Sic1 (ref. 15) (Fig. 1a).Sae2 contains three potential CDK phosphorylation sites ( Fig. 1b and Supplementary Fig. 1); two of these-Ser 267 and Ser 134-received the highest scores for predicted phosphorylation sites in the protein (Supplementary Table 1). Ser 267 maps to the Sae2 region most highly conserved with its non-yeast orthologues (Fig. 1b), which include human CtIP, Caenorhabditis elegans Com1 and Arabidopsis thaliana Com1 (refs 16-18). To address the possible function(s) of Ser 267 and other potential target site...