Mitotic double-strand break repair at the mating type locus in Saccharomyces cerevisiae was analyzed in thermosensitive strains, carrying essential replication factors. Mutations in either leading strand polymerase, polymerase delta or epsilon, indicate that either polymerase can function in MAT switching, but their processivity cofactors, PCNA and RF-C, are essential, as demonstrated by the complete absence of the initial strand invasion intermediate step, and of final recombinant product formation at the non-permissive temperature. Mutants of the lagging strand machinery, DNA polymerase alpha ( pol1-17 ), DNA primase ( pri2-1 ) and Rad27 ( rad27 ), greatly inhibit strand invasion and product formation, compared to wild type levels. This lagging strand requirement remains even when the substrate donor for repair shares perfect homology with both sides of the cut MAT locus. We propose a model in which a double-strand break (DSB) at MAT is converted to a replication fork. When the lagging strand machinery is inactivated, the cell repairs the DSB very inefficiently. Therefore, the model for double-strand break repair, proposed by Szostak et al ., invoking only leading strand repair synthesis, is not the preferred mechanism in our system.
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