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Double Holliday junctions are intermediates of DNA break repair

Abstract

Repair of DNA double-strand breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumour suppression. However, despite its importance, the molecular intermediates of mitotic DSB repair remain undefined. The double Holliday junction (DHJ), presupposed to be the central intermediate for more than 25 years1, has only been identified during meiotic recombination2. Moreover, evidence has accumulated for alternative, DHJ-independent mechanisms3,4,5,6, raising the possibility that DHJs are not formed during DSB repair in mitotically cycling cells. Here we identify intermediates of DSB repair by using a budding-yeast assay system designed to mimic physiological DSB repair. This system uses diploid cells and provides the possibility for allelic recombination either between sister chromatids or between homologues, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homologue joint molecule (JM) intermediates whose strand composition and size are identical to those of the canonical DHJ structures observed in meiosis2. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologues. Moreover, JMs seem to represent a minor pathway of DSB repair in mitotic cells, being detected at about tenfold lower levels (per DSB) than during meiotic recombination. Thus, although DHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.

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Figure 1: Inducible DSB repair system.
Figure 2: Detection of JMs during DSB repair.
Figure 3: Analysis of JM composition.
Figure 4: Temporal analysis of JMs in wild-type and sgs1 cells.

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Acknowledgements

We thank R. Schiestl for plasmid pWY203, and W. Heyer for critical reading of the manuscript. This work was supported by National Institutes of Health (NIH) National Institute of General Medical Sciences grants GM025326 to N.K. and GM074223 to N.H.

Author Contributions M.B. performed the experiments, assisted by S.D.O. and N.T. M.B., N.K. and N.H. designed the experiments, discussed the results and prepared the manuscript.

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Correspondence to Neil Hunter.

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Bzymek, M., Thayer, N., Oh, S. et al. Double Holliday junctions are intermediates of DNA break repair. Nature 464, 937–941 (2010). https://doi.org/10.1038/nature08868

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