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Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis

Abstract

The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading- and lagging-strand DNA synthesis. In contrast, ssDNA gaps behind forks, which are prevalent on damaged templates, result from Mre11-dependent degradation of newly synthesized DNA strands and are suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct roles for Rad51 at replication forks, demonstrating that Rad51 protects newly synthesized DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.

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Figure 1: Rad51 binding to undamaged and damaged chromatin during DNA replication.
Figure 2: Rad51 and PCNA modifications in DNA replication and ssDNA gap accumulation.
Figure 3: Rad51 is required to prevent replication fork uncoupling and ssDNA accumulation on damaged and undamaged templates.
Figure 4: Accumulation of ssDNA gaps in the absence of Rad52 and Rad51 in S. cerevisiae.
Figure 5: Rad51 protects nascent strand DNA from Mre11-dependent degradation.
Figure 6: A model for possible roles of Rad51 during DNA replication.

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Acknowledgements

We thank S. West, S. Boulton and members of the genome stability lab for their insightful comments, H. Mahbubani, J. Kirk for technical support with X. laevis and the Center for Microscopy and Image Analysis of the University of Zurich for technical assistance with electron microscopy. This work was funded by Cancer Research UK. V.C. is also supported by the European Research Council start-up grant (206281), the Lister Institute of Preventive Medicine and the European Molecular Biology Organization (EMBO) Young Investigator Program. M.L. and A.R.C. are supported by the Swiss National Science Foundation grant (PP00A-114922). A.R.C. was also supported by an EMBO short-term fellowship. Recombinant human Rad51 proteins and the cDNA fragment encoding human BRC4 (residues 1511–1579 of BRCA2) cloned into pDONR221 were provided by F. Esashi (Oxford University). The pET28-based expression vectors of wild-type and mutant (K164R) X. laevis PCNA and the pET21-based expression vector of human p27 were provided by H. Ulrich (Cancer Research UK) and T. Hunt (Cancer Research UK), respectively. Mirin was provided by J. Gautier (Columbia University). Antibodies to Mcm2, Cdc45, Psf2 and Pol ɛ p60 subunit were provided by H. Takisawa (Osaka University); antibodies to Pol δ p125 subunit and Pol η were provided by S. Waga (Japan Women's University) and M. Akiyama (Nara Institute of Technology).

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Y.H. and A.R.C. carried out experiments; M.L. and V.C. conceived experiments and wrote the manuscript.

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Correspondence to Massimo Lopes or Vincenzo Costanzo.

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Hashimoto, Y., Ray Chaudhuri, A., Lopes, M. et al. Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat Struct Mol Biol 17, 1305–1311 (2010). https://doi.org/10.1038/nsmb.1927

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