Double Holliday junctions are intermediates of DNA break repair

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Abstract
In meiotic cells, it is well established that the paired homologues are joined by a set of crossovers known as a double Holliday junction (DHJ). Whether DHJs form during mitotic recombination has been unclear, since mitotic cells possess alternative repair pathways that would not require DHJ formation. Bzymek et al. now demonstrate that mitotic and meiotic cells form similar DHJs, but that the levels in mitotic cells are approximately 10-fold lower, and show a preference for joints between sister chromatids rather than homologues. Consequently in mitotic cells non-crossover outcomes are favoured. In meiotic cells paired homologues are joined by a set of crossovers known as a double Holliday junction (DHJ). Whether DHJs form during mitotic recombination has been unclear, as mitotic cells possess alternative repair pathways that would not require DHJ formation. Here it is demonstrated that mitotic and meiotic cells form similar DHJs, but that the levels in mitotic cells are approximately 10–fold lower, and show a preference for joints between sister chromatids rather than homologues. Consequently, in mitotic cells non–crossover outcomes are favoured. 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.