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RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks

Nature Structural & Molecular Biology volume 19, pages 17–24 (2012)Cite this article

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Abstract

In higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45–MCM–GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ɛ onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.

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Figure 1: RAD51 is required for DNA replication in the presence of forks collapsed by a single-strand break in the template.
Figure 2: RAD51 is required for stable chromatin association of fork proteins in the presence of template breakage.
Figure 3: RAD51 is required for origin-independent fork restart and reloading of replisome components after fork collapse.
Figure 4: MRE11 nuclease activity is required for DNA replication upon fork collapse.
Figure 5: The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse.
Figure 6: A model of replication fork collapse and restart.

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Acknowledgements

We are grateful to J. Haber and J. Lydeard for sharing unpublished results on PCNA BIR–defective mutants. We thank members of the genome stability lab for their insightful comments. We thank H. Mahbubani and J. Gannon for technical support with X. laevis. This work was funded by Cancer Research UK. V.C. is also supported by the European Research Council (ERC) startup grant (206281), the Lister Institute of Preventive Medicine and the European Molecular Biology Organization (EMBO) Young Investigator Program (YIP).

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  1. Genome Stability Unit, Clare Hall Laboratories, London Research Institute, South Mimms, Hertfordshire, UK

    Yoshitami Hashimoto, Fabio Puddu & Vincenzo Costanzo

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  1. Yoshitami Hashimoto
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  2. Fabio Puddu
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Y.H. and F.P. performed experiments. Y.H. and V.C. analyzed the results and wrote the manuscript.

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Correspondence to Vincenzo Costanzo.

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Hashimoto, Y., Puddu, F. & Costanzo, V. RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks. Nat Struct Mol Biol 19, 17–24 (2012). https://doi.org/10.1038/nsmb.2177

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