| Double-strand breaks can initiate homologous and nonhomologous pathways of repair. This thesis focuses on HO-induced DSB repair involving bacterial and yeast homologous inverted repeats in S. cerevisiae. This system allowed for in vivo physical monitoring of the kinetics of repair and of the final levels of gene conversions associated with or without crossovers.; First we demonstrated that the nucleotide excision repair proteins, Rad1 and Rad10, and the mismatch repair proteins, Msh2 and Msh3, act on the removal of 3′ nonhomologous tails from recombination intermediates during DSB repair. This was done by monitoring repair in a centromeric plasmid carrying inverted LacZ repeats, where the double-strand break was flanked by increasing amounts of nonhomologous sequences. We also identified a RAD1- and MSH2-independent pathway able to process a single nonhomologous end. When the nonhomologous end was closer to the 3′ end of LacZ, a reduction in the proportion of gene conversions associated with crossovers occurred in the absence of RAD1 or MSH2. Finally, an uncoupling was observed between gene conversions and crossovers, dependent on the absence of the Rad1/Rad10 and Msh2/Msh3 proteins and on the presence of one homologous tail. These data support the synthesis-dependent strand annealing pathway as a major mechanism of DSB repair in S. cerevisiae.; Intrachromosomal DSB repair poises a barrier against gene conversions associated with crossovers. Plasmids are structurally less constrained for invasion and stabilization of intermediates and should allow for higher levels of crossovers. While plasmids containing inverted LacZ and LEU2 repeats repaired respectively 43% and 35% of events via gene conversions associated with crossovers, a similar plasmid carrying inverted MATα repeats, maintained the constrain against crossovers (4% crossovers) indicating sequence-specific effects on crossover regulation involving the MAT gene.; Finally, ressection of DSB ends is affected by the absence of RAD1 or MSH2. Physical and genetic assays have led us to propose that in the absence of these proteins, Exonuclease I affects loading of the more processive MRE11/XRS2/ RAD50 complex affecting the kinetics of repair. |
