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The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks

Abstract

Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair.

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Figure 1: Analysis of DSB formation induced by DNA replication inhibitors in wild-type and Mus81-deficient mouse ES cells.
Figure 2: Time course of DSB formation and cell-cycle analysis of wild-type and Mus81−/− ES cells treated with hydroxurea.
Figure 3: Time course of DSB formation and cell-cycle analysis of wild-type and Mus81−/− ES cells in response to aphidicolin.
Figure 4: Sensitivity of wild-type and Mus81−/− ES cells to hydroxyurea, aphidicolin and ionizing radiation.
Figure 5: Recovery of DNA replication after treatment with hydroxyurea.
Figure 6: Response of Rad54−/− ES cells to hydroxyurea.
Figure 7: Rad51 focus formation and colocalization in response to hydroxyurea.
Figure 8: Chromosomal aberrations after treatment with hydroxyurea in wild-type and Mus81−/− ES cells.

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Acknowledgements

We thank C. Beerens for technical help. This work was supported by grants from the Dutch Cancer Society (KWF), the Netherlands Organization for Scientific Research (NWO), the European Commission (IP 512113) and by Cancer Research UK.

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Authors and Affiliations

Authors

Contributions

K.H. generated the reagents and designed and carried out the experiments. M.B. carried out a number of the PFGE experiments and designed and carried out the FACS experiments. S.L.D. analyzed the DNA fiber experiments. E.v.D. and H.B.B. carried out and analyzed the chromosomal aberration experiments. H.O. carried out the Rad54 and Rad51 localization experiments. A.M. and J.E. were involved in generating ES cells. I.D.H. supervised the fiber experiments. R.K. advised on the design of the experiments. K.H., I.D.H. and R.K. were responsible for the preparation of the manuscript.

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Correspondence to Roland Kanaar.

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Hanada, K., Budzowska, M., Davies, S. et al. The structure-specific endonuclease Mus81 contributes to replication restart by generating double-strand DNA breaks. Nat Struct Mol Biol 14, 1096–1104 (2007). https://doi.org/10.1038/nsmb1313

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