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Cell-cycle coordination between DNA replication and recombination revealed by a vertebrate N-end rule degron-Rad51

Nature Structural & Molecular Biology volume 15, pages 1049–1058 (2008)Cite this article

Abstract

Coordination of homologous DNA recombination (HDR) with DNA replication maintains the fidelity of cell division. Exploiting Varshavsky's N-end rule to create a thermosensitive degron for conditional genetics in an avian cell line, we confirm that inactivation of the essential HDR enzyme Rad51 in a single cell cycle does not stop replicative DNA synthesis but, instead, causes G2 arrest. Rad51 complementation after the completion of replication overcomes this block, suggesting that HDR becomes necessary in G2. Indeed, DNA structures that bind activated replication protein A accumulate during the S phase, to be preferentially resolved during G2 by a Rad51-dependent mechanism. Breaks affecting a single chromatid predominate after the first cell cycle without Rad51, subsequently evolving into isochromatid lesions. We suggest a model for the vertebrate cell cycle in which HDR during the G2 phase is separated from DNA replication in S phase and chromosome segregation in M.

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Figure 1: A thermosensitive conditional N-end rule degron in DT40 cells.
Figure 2: Degron-RAD51 supports cell viability.
Figure 3: Degron-RAD51 supports sister-chromatid exchange (SCE).
Figure 4: RAD51 is dispensable for replicative DNA synthesis but becomes essential after replication for mitotic entry.
Figure 5: RAD51 complementation after the completion of replicative DNA synthesis suffices to prevent G2 arrest.
Figure 6: ssDNA structures marked by anti–p-RPA staining accumulate in the first cell cycle without RAD51.
Figure 7: ssDNA structures are preferentially resolved during G2 by a RAD51-dependent mechanism.
Figure 8: ssDNA foci accumulate during unchallenged replication in wild-type cells and colocalize with RAD51 foci preferentially during G2.

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Acknowledgements

We are grateful to E. Nigg (Max Planck Institute, Martinsreid, Germany) for providing anti–cyclin B, K. Labib (PICR, Manchester) for providing pKL187 and S. Takeda (Kyoto University, Japan) for providing the Rad51−/−(tetRAD51) DT40 strain. We thank N. Ayoub for help in establishing the degron-EGFP system in DT40, E. Rajendra for technical help with the DT40 experiments and critical review of the manuscript, and members of our laboratory for helpful discussions. X.S. is supported by a studentship from A*STAR, Singapore, and J.A.B. by a Wellcome Trust grant to A.R.V. Work in A.R.V.'s laboratory is supported by the UK Medical Research Council.

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  1. The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 0XZ, UK

    Xinyi Su, Juan A Bernal & Ashok R Venkitaraman

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Contributions

X.S. and J.A.B. together performed the experiments, with J.A.B. as the primary contributor to Figures 4c and 5c and Supplementary Figure 3, and X.S. as the primary contributor to the remaining figures. J.A.B. was instrumental in optimizing the experimental conditions for the use of N-end degrons in DT40 cells and provided guidance to X.S. X.S., J.A.B. and A.R.V. interpreted the data. X.S. and J.A.B. prepared the figures. A.R.V. supervised the work and wrote the paper with the help of X.S. and J.A.B.

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Correspondence to Ashok R Venkitaraman.

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Su, X., Bernal, J. & Venkitaraman, A. Cell-cycle coordination between DNA replication and recombination revealed by a vertebrate N-end rule degron-Rad51. Nat Struct Mol Biol 15, 1049–1058 (2008). https://doi.org/10.1038/nsmb.1490

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