Evidence for Exo1-protected DNA nicks directing crossover formation in meiosis

Most crossovers that form between homologous chromosomes during meiosis in budding yeast result from the biased resolution of double Holliday Junction (dHJ) intermediates. This resolution occurs in steps that involve the Rad2/XPG nuclease Exo1 and the Mlh1-Mlh3 mismatch repair endonuclease, but little is known at the molecular level about how biased resolution occurs. Here we show that Exo1 acts in a pro-crossover role in meiosis by protecting DNA nicks from ligation. Through the analysis of a large set of exo1 alleles, we found that while the catalytic activity of Exo1 appears dispensable for its pro-crossover role, structural elementsrequired for direct DNA interactions, including binding and bending of DNA during nick/flap recognition, are crucial. Consistent with these observations, meiotic expression of the Rad2/XPG family member Rad27 partially rescued the crossover defect in exo1 null mutants, and meiotic overexpression of Cdc9 ligase specifically reduced the crossover levels of exo1 DNA binding mutants to levels approaching the exo1 null. In addition, our work identified a role for Exo1 in crossover interference that does not appear to be explained by defects inresection activity. Together, these studies provide experimental evidence for Exo1-protected nicks being critical for the formation of meiotic crossovers.