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.
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We thank T. Paull and J. Falck for providing reagents, and P. Huertas, A. Meier, K. Dry, K. Miller and Y. Pommier for advice and critical reading of the manuscript. This study was supported by Cancer Research UK, the EU and a Swiss National Science Foundation fellowship for advanced researcher (A.A.S). Research in the S.P.J. laboratory is made possible by core infrastructure funding from Cancer Research UK and the Wellcome Trust. C.L., J.L., M.M. and J.B. were supported by grants from the Danish Cancer Society, Danish National Research Foundation, EU (DNA Repair), and the John and Birthe Meyer Foundation and the Czech Ministry of Education. Research in the laboratory of R.B. is supported by the National Institute of Health and S.F. is supported by a fellowship from the New York State Breast Cancer Research Fund.
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 homologous recombination 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.
The authors declare no competing financial interests.
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Sartori, A., Lukas, C., Coates, J. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007). https://doi.org/10.1038/nature06337
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