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Human Ctip Promotes DNA End Resection

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Human Ctip Promotes DNA End Resection doi:10.1038/nature06337 ARTICLES Human CtIP promotes DNA end resection Alessandro A. Sartori1, Claudia Lukas2, Julia Coates1, Martin Mistrik2, Shuang Fu3, Jiri Bartek2, Richard Baer3, Jiri Lukas2 & Stephen P. Jackson1 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 signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the 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 homologous recombination. Finally, we reveal that CtIP has 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 signalling and homologous recombination. DSBs are highly cytotoxic lesions induced by ionizing radiation and exposure. Short interfering (si)RNA-mediated depletion of CtIP certain anti-cancer drugs. They also arise when replication forks caused hypersensitivity towards the topoisomerase I inhibitor camp- encounter other lesions and are generated as intermediates during tothecin and the topoisomerase II inhibitor etoposide (Fig. 1a, b) but meiotic recombination1. Cells possess two main DSB repair mechan- did not alter cell-cycle distribution profiles and only modestly isms: non-homologous end-joining and homologous recombi- decreased the proportion of replicating cells (Supplementary Fig. nation. Whereas non-homologous end-joining predominates in 1a). Furthermore—and consistent with the major cytotoxic lesions G0/G1 and is error-prone, homologous recombination is restricted caused by camptothecin and etoposide being DSBs arising during to S and G2, when sister chromatids allow faithful repair2–4. S phase23—the effects of CtIP depletion were largely abrogated by Homologous recombination is initiated by resection of DSBs to treating cells with the DNA-replication inhibitor aphidicolin generate single-stranded DNA (ssDNA) that binds RPA. A ssDNA– (Fig. 1a, b). These data therefore suggested that CtIP depletion RAD51 nucleoprotein filament then forms to initiate strand inva- impairs cell survival when DSBs are generated during S phase. Also, sion. ssDNA–RPA also recruits ATR, triggering ATR-dependent because DSBs generated in S phase by camptothecin are repaired by checkpoint signalling by the protein kinase CHK1 (also known as homologous recombination24, these results suggested that CtIP pro- CEHK1) (ref. 5). Because DSB resection is largely restricted to S and motes homologous recombination. Congruent with CtIP being G2, both homologous recombination and checkpoint signalling by unnecessary for non-homologous end-joining, its depletion caused ATR are subject to cell-cycle control6–8. only weak hypersensitivity towards bleocin, which generates DSBs at A factor implicated in DSB resection is the MRE11–RAD50–NBS1/ all cell-cycle stages (Fig. 1c, and Supplementary Fig. 1b). NBN (MRN) complex, which binds DNA ends, possesses exo- and We next assessed the impact of CtIP on phosphorylations endo-nuclease activities and functions in triggering DNA-damage mediated by the protein kinases ATM and ATR. In response to checkpoints9,10. Here, we show that human CtIP physically and func- DSBs, ATM phosphorylates the checkpoint kinase CHK2 (also tionally interacts with MRN. CtIP—initially identified as an interactor known as CHEK2) and histone H2AX (also known as H2AFX; gen- for CtBP11 and the tumour suppressor proteins RB1 (ref. 12) and erating a protein species termed cH2AX)25,26. In contrast, ATR BRCA1 (refs 13, 14)—is recruited to DNA damage and complexes activation by ssDNA elicits CHK1 phosphorylation5. As shown in with BRCA1 to control the G2/M DNA-damage checkpoint15–17. We Fig. 1d, camptothecin triggered cH2AX formation and the genera- reveal that CtIP also promotes ATR activation and homologous tion of a slower-migrating, hyper-phosphorylated species of CtIP recombination by cooperating with MRN to mediate DSB resection. (CtIP hyper-phosphorylation was demonstrated by phosphatase On the basis of these findings and our identification of sequence treatments and a phospho-specific antibody; Supplementary Fig. homologies between CtIP and Sae2—a protein implicated in mitotic 1c, d). Furthermore, both CtIP and H2AX phosphorylation were pre- and meiotic DSB processing in Saccharomyces cerevisiae18–22—we con- vented when aphidicolin was co-administered with camptothecin. clude that these proteins are functional homologues. Etoposide also triggered modification of H2AX and CtIP; however, in this case, CtIP phosphorylation but not H2AX phosphorylation CtIP affects cellular responses to DSBs was abrogated by aphidicolin (Fig. 1e). These data are in accord with To investigate CtIP function, we examined how its depletion affected etoposide being able to generate DSBs throughout the cell cycle, and clonogenic survival of human U2OS cells, following their treatment suggest that CtIP phosphorylation occurs most effectively in S phase. with DNA-damaging agents. To circumvent possible effects arising CtIP depletion did not affect H2AX phosphorylation triggered by from CtIP’s involvement in controlling cell-cycle progression, we either camptothecin or etoposide, revealing that CtIP is not required employed acute (1 h) drug treatments rather than continuous drug for DNA-lesion generation by these drugs (Fig. 1d, e). Similarly, 1The Wellcome Trust and Cancer Research UK Gurdon Institute, and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK. 2Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. 3Institute for Cancer Genetics, Department of Pathology, Columbia University, New York, New York 10032, USA. 1 © 2007 Nature Publishing Group ARTICLES NATURE camptothecin-induced phosphorylations of CHK2 and SMC1 were was the case, CtIP might influence hyper-phosphorylation of the unaffected by CtIP depletion, indicating that ATM activation still amino-terminal region of RPA2 by ATM, ATR and DNA-PK (also occurs when CtIP is absent (Fig. 1f, and data not shown). In contrast, known as PRKDC) in the context of replication-associated DSBs28,29. CtIP depletion diminished camptothecin-induced CHK1 phosphory- Indeed, camptothecin-induced RPA2 phosphorylation—as measured lation on Ser 345 and Ser 317 (Fig. 1f, and data not shown). Notably, by a shift in RPA2 electrophoretic mobility and by phospho-specific CtIP depletion markedly impaired CHK1 phosphorylation at late antibodies against modified RPA2 serines 4 and 8—was markedly time-points but not at early time-points after continuous camptothe- impaired by CtIP depletion, whereas DSB formation, as measured cin exposure (Supplementary Fig. 1e). This suggested that CtIP is not by cH2AX formation, was unaffected (Fig. 1g). Similarly, CtIP required for initial CHK1 phosphorylation triggered by replication depletion impaired RPA hyper-phosphorylation but not CHK2 or stress induced by trapped DNA topoisomerase I molecules, but is SMC1 phosphorylation in response to etoposide (data not shown). needed for signalling of camptothecin-induced replication-dependent Together, these data indicated that CtIP is required neither for DSB DSBs, which, as for ionizing-radiation-induced DSBs7,27, might generation by topoisomerase inhibitors nor for DSB detection and require resection to elicit ATR activation. We reasoned that if this signalling by ATM. Instead, CtIP is specifically needed for efficient activation and/or propagation of ATR-mediated signalling. a d 100 siRNA: Luc CtIP-1 CtIP-2 CtIP promotes ATR recruitment in S/G2 Aphidicolin: – – + – – + – – + Etoposide: – + + – + + – + + When we generated DSB-containing tracks in human U2OS cell 80 30 CtIP * nuclei by laser micro-irradiation , CtIP was recruited to the damage γH2AX with kinetics slower than that of cH2AX formation (Fig. 2a). 60 Tubulin a 40 siLuc siLuc + aph. Survival (%) siCtlP-1 20 siCtlP-1 + aph. H2AX siCtlP-2 e γ siCtlP-2 + aph. siRNA: Luc CtIP-1 0 0 0.01 0.1 1 Aphidicolin: – – + – – + Etoposide: – + + – + + Camptothecin (µM) * CtIP CtlP b γH2AX 100 Tubulin 1 2 3 4 6 12 Time after laser micro-irradiation (min) 80 γH2AX/ b cyclin A CtIP c GFP–CtIP Merge 60 S/G2 f 40 siRNA: Luc CtIP-1 siLuc G1 H2AX Survival (%) Camptothecin: – + – + γ siLuc + aph. 20 * siCtlP-1 CtIP siCtlP-1 + aph. 0 MRE11 0 2 5 CHK2 pT68 Etoposide (µM) CHK1 pS345 RPA2 c Merge 100 d 70 H2AX GFP–ATR 80 60 Ctrl siRNA e γ g CtlP siRNA 50 60 siRNA: Luc CtIP-1 40 Camptothecin: – 15 60 – 15 60 40 * Ctrl siRNA CtIP 30 Survival (%) siLuc 20 20 RPA2pS4/S8 siCtlP * RPA2 Proportion of cells (%) 10 siXRCC4 siRNA 0 γH2AX 0 0 2 5 10 Strong Weak None CtlP –1 H2A Bleocin (µg ml ) Relative intensity of GFP–ATR in micro-irradiated area (15 min) Figure 1 | CtIP depletion causes hypersensitivity to DSB-inducing agents. a–c, CtIP downregulation causes replication-dependent camptothecin and Figure 2 | CtIP associates with sites of DNA damage
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