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Polymerase δ replicates both strands after homologous recombination–dependent fork restart

Nature Structural & Molecular Biology volume 22, pages 932–938 (2015)Cite this article

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Abstract

To maintain genetic stability, DNA must be replicated only once per cell cycle, and replication must be completed even when individual replication forks are inactivated. Because fork inactivation is common, passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination–dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via homologous recombination–restarted replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis because HoRReR is more error prone than canonical replication. This increased error rate implies the HoRReR mechanism is distinct from that of a canonical fork. Here we demonstrate, in Schizosaccharomyces pombe, that a DNA sequence duplicated by HoRReR during S phase is replicated semiconservatively, but both the leading and lagging strands are synthesized by DNA polymerase δ.

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Figure 1: Polδ, but not Polɛ, participates in bulk DNA synthesis after HR restart.
Figure 2: Polδ usage is relatively uniform, and Polα usage decreases.
Figure 3: HR-restarted replication is semiconservative.
Figure 4: Instability resulting from HR-restarted replication is not intrinsic to leading-strand synthesis by Polδ.
Figure 5: Polδ usage correlates with fragile sites.

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Acknowledgements

This work was supported by Project Z01 ES065070 (T.A.K.) from the Division of Intramural Research of the US National Institutes of Health, by Medical Research Council (UK) grants G0801078 and G1100074 (A.M.C.) and by European Research Council grant 268788-SMI-DDR (A.M.C.). We thank H. Masukata (Osaka University) for the cdc20::hphMX6-Pnmt1-cdc20CTD strain.

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Authors and Affiliations

  1. Genome Damage and Stability Centre, University of Sussex, Brighton, UK

    Izumi Miyabe, Ken'Ichi Mizuno, Andrea Keszthelyi, Meliti Skouteri, Saed Mohebi, Johanne M Murray & Antony M Carr

  2. Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan

    Yasukazu Daigaku

  3. Department of Health and Human Services, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA

    Thomas A Kunkel

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Contributions

A.M.C. conceived the study. A.M.C., T.A.K., I.M. and J.M.M. designed the experimental approach. I.M., K'I.M., S.M., Y.D., A.K. and M.S. performed experiments and interpreted data. I.M. and A.M.C. wrote the manuscript. T.A.K. and J.M.M. edited the manuscript.

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Correspondence to Antony M Carr.

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Miyabe, I., Mizuno, K., Keszthelyi, A. et al. Polymerase δ replicates both strands after homologous recombination–dependent fork restart. Nat Struct Mol Biol 22, 932–938 (2015). https://doi.org/10.1038/nsmb.3100

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