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Cell cycle regulation of DNA double-strand break end resection by Cdk1-dependent Dna2 phosphorylation

Nature Structural & Molecular Biology volume 18, pages 10151019 (2011) | Download Citation

Abstract

DNA recombination pathways are regulated by the cell cycle to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5′ strand resection at DNA double-strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11–Rad50–Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase–topoisomerase complex Sgs1–Top3–Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae, we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting cross-talk between Dna2- and Exo1-dependent resection pathways.

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Acknowledgements

We thank N. Lyons and D. Morgan (University of California, San Francisco) for providing Cdk1 kinase and valuable advice. This work was supported by US National Institutes of Health (NIH) grants GM080600 and 3R01GM080600 to G.I., RO1GM57814 and RO1ES07061 to P.S., and GM071011 and 3R01 GM071011 to S.E.L.

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Affiliations

  1. Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

    • Xuefeng Chen
    • , Woo-Hyun Chung
    • , Zhu Zhu
    • , Alma Papusha
    •  & Grzegorz Ira
  2. Department of Molecular Biophysics & Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Hengyao Niu
    •  & Patrick Sung
  3. Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

    • Eun Yong Shim
    •  & Sang Eun Lee

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Contributions

X.C. constructed most of the strains and plasmids, analyzed Dna2 phosphorylation in cells, carried out fluorescence microscopy and ChIP experiments. W.-H.C. and A.P. analyzed resection in mutants, and H.N. purified proteins and conducted all the in vitro experiments. Z.Z. constructed the initial plasmids carrying dna2 mutant alleles. X.C., H.N., P.S. and G.I. designed the experiments, analyzed the data and wrote the manuscript. E.Y.S. and S.E.L. did the ChIP assay for Sgs1-13 × Myc.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Patrick Sung or Grzegorz Ira.

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DOI

https://doi.org/10.1038/nsmb.2105

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