Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

Abstract Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication/recombination intermediates. Here, we employed a biochemical approach to demonstrate that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we confirm that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.