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CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle

Abstract

The repair of DNA double-strand breaks (DSBs) is tightly regulated during the cell cycle. In G1 phase, the absence of a sister chromatid means that repair of DSBs occurs through non-homologous end-joining or microhomology-mediated end-joining (MMEJ)1. These pathways often involve loss of DNA sequences at the break site and are therefore error-prone. In late S and G2 phases, even though DNA end-joining pathways remain functional2, there is an increase in repair of DSBs by homologous recombination, which is mostly error-free3,4. Consequently, the relative contribution of these different pathways to DSB repair in the cell cycle has a large influence on the maintenance of genetic integrity. It has remained unknown how DSBs are directed for repair by different, potentially competing, repair pathways. Here we identify a role for CtIP (also known as RBBP8) in this process in the avian B-cell line DT40. We establish that CtIP is required not only for repair of DSBs by homologous recombination in S/G2 phase but also for MMEJ in G1. The function of CtIP in homologous recombination, but not MMEJ, is dependent on the phosphorylation of serine residue 327 and recruitment of BRCA1. Cells expressing CtIP protein that cannot be phosphorylated at serine 327 are specifically defective in homologous recombination and have a decreased level of single-stranded DNA after DNA damage, whereas MMEJ remains unaffected. Our data support a model in which phosphorylation of serine 327 of CtIP as cells enter S phase and the recruitment of BRCA1 functions as a molecular switch to shift the balance of DSB repair from error-prone DNA end-joining to error-free homologous recombination.

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Figure 1: Sensitivity of CtIP -/-/- mutant cells to DNA-damaging agents.
Figure 2: CtIP -/-/- mutant cells are sensitive to X-rays in both G1 and S/G2 phases of the cell cycle.
Figure 3: CtIP -/-/- mutant cells are defective for homologous recombination and MMEJ.
Figure 4: Phosphorylation of serine 327 is required for generation of ssDNA in DT40 cells.

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Acknowledgements

The authors would like to thank M. Jasin for gifts of DR–GFP and pHPRT-SSA-GFP, S. Takeda for the gift of KU70 DT40, and J. Di Noia for DT40 DTDR-17. We would also like to thank our colleagues C. Rada and J. Sale for comments and suggestions during the preparation of this manuscript. M.H.Y. is a Milstein Student of the Darwin Trust, Edinburgh, Scotland.

Author Contributions All experiments were performed by M.H.Y. and were conceived by M.H.Y. and K.H. K.H. and M.H.Y. wrote the paper.

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Yun, M., Hiom, K. CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle. Nature 459, 460–463 (2009). https://doi.org/10.1038/nature07955

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