Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae

A plasmid gap repair assay was used to assess the role of three known nucle ases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay , alterations in end processing or branch migration are reflected by the fr equency of co-conversion of a chromosomal marker 200 bp from the gap. Gap r epair associated with crossing over results in integration at the homologou s chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3 - to 10-fold and conversion tracts were shorter than in the wild-type strai n, consistent with a role for this nuclease in processing double-strand bre aks. However, conversion tracts were longer in a strain containing the nucl ease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 5-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was incre ased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutan t level.