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...

Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing of ICLs requires the Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5'-->3' exonuclease and a structure-specific endonuclease that preferentially incises 5' flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.

The bacterial pathogen Helicobacter pylori chronically infects the human gastric mucosa and is the leading risk factor for the development of gastric cancer. The molecular mechanisms of H. pyloriassociated gastric carcinogenesis remain ill defined. In this study, we examined the possibility that H. pylori directly compromises the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs) in primary and transformed murine and human epithelial and mesenchymal cells. The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA by pulse field gel electrophoresis and by high-magnification microscopy of metaphase chromosomes. Bacterial adhesion (e.g., via blood group antigenbinding adhesin) is required to induce DSBs; in contrast, the H. pylori virulence factors vacuolating cytotoxin A, γ-glutamyl transpeptidase, and the cytotoxin-associated gene (Cag) pathogenicity island are dispensable for DSB induction. The DNA discontinuities trigger a damage-signaling and repair response involving the sequential ataxia telangiectasia mutated (ATM)-dependent recruitment of repair factors-p53-binding protein (53BP1) and mediator of DNA damage checkpoint protein 1 (MDC1)-and histone H2A variant X (H2AX) phosphorylation. Although most breaks are repaired efficiently upon termination of the infection, we observe that prolonged active infection leads to saturation of cellular repair capabilities. In summary, we conclude that DNA damage followed by potentially imprecise repair is consistent with the carcinogenic properties of H. pylori and with its mutagenic properties in vitro and in vivo and may contribute to the genetic instability and frequent chromosomal aberrations that are a hallmark of gastric cancer.

End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombinationrelies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1). In this study, we show that the localization of EXO1 to DSBs depends on both CtIP and MRN. We also establish that CtIP interacts with EXO1 and restrains its exonucleolytic activity in vitro. Finally, we show that on exposure to camptothecin, depletion of EXO1 in CtIPdeficient cells increases the frequency of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity.

Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer.

The resolution of DNA interstrand crosslinks (ICLs) requires a complex interplay between several processes of DNA metabolism, including the Fanconi anemia (FA) pathway and homologous recombination (HR). FANCD2 monoubiquitination and CtIP-dependent DNA-end resection represent key events in FA and HR activation, respectively, but very little is known about their functional relationship. Here, we show that CtIP physically interacts with both FANCD2 and ubiquitin and that monoubiquitinated FANCD2 tethers CtIP to damaged chromatin, which helps channel DNA double-strand breaks generated during ICL processing into the HR pathway. Consequently, CtIP mutants defective in FANCD2 binding fail to associate with damaged chromatin, which leads to increased levels of nonhomologous end-joining activity and ICL hypersensitivity. Interestingly, we also observe that CtIP depletion aggravates the genomic instability in FANCD2-deficient cells. Thus, our data indicate that FANCD2 primes CtIP-dependent resection during HR after ICL induction but that CtIP helps prevent illegitimate recombination in FA cells.