Analysis of the processing of complex recombination intermediates by structure-selective endonucleases

Abstract

DNA repair by homologous recombination involves the formation of branched intermediates that can constitute a source of genome instability when untimely processed. To prevent these structures from interfering with chromosome segregation, cells rely on the action of the structure-selective endonucleases Mus81-Mms4 and Yen1. Here, we employed a biochemical approach to show that both endonucleases can process the central intermediate of this pathway, the displacement loop (D-loop). Using synthetic and enzymatically reconstituted D-loops, we mapped their incision sites and recapitulated for the first time the formation of a half-crossover precursor in vitro. This implies that the concurrent action of Mus81-Mms4 and Yen1 on a D-loop may lead to the generation of complex chromosomal rearrangements.