| A detailed account of the molecular events during DSB induced recombination and the role of genes important for recombinational repair is described herein. HO endonuclease-induced double strand breaks in Saccharomyces cerevisiae recombine by two distinct and competing pathways. In direct repeat constructs, in which one repeat is interrupted by an HO cut site, gene conversions are produced by gap-repair while deletions are produced by single-strand annealing. These two pathways are independently modulated by changes in the substrate structure, cell-cycle conditions and by recombination deficient mutations.; Consistent with predictions of the single-strand annealing mechanism, deletion formation is dependent on the time in which complementary regions become single-stranded. Increasing the distance between the repeats leads to a delay in deletion formation. Gap repair processes are independent of distance but are reduced in rad52 mutants and in G1-arrested cells. These two pathways compete for a common intermediate as when either pathway is slowed, the proportion of products from the other pathway increases.; UV-sensitive rad1 deletion strains repair chromosomal DSBs where one or both ends of the break contain homologous sequences. However, they cannot complete either gene conversion or deletion formation when the ends of the cut DNA contain 60 bp of non-homology that must first be removed before homologous recombination can proceed. The requirement to remove the non-homology is bypassed and gap-repair and single-strand annealing occur when the ends of the break are made homologous to donor sequences. RAD1 has been implicated in the endonucleolytic excision of thymidine dimers, which suggests that it also acts as an endonuclease in removing non-homology from the ends of recombining DNA.; Both gap-repair and single-strand annealing occur independently of the rad51 gene product which may encode a strand exchange protein. However, the kinetics of gene conversion formation are greatly slowed. In contrast, the site specific mating type switch requires the RAD51 gene product. These results suggest that the recombination protein components required for specific gap-repair processes are limited to specific DNA substrates. |
