Use of a unique reporter cassette to examine 5´ to 3´ resection at a site specific DNA double-strand break during meiosis
Meiotic recombination in Saccharomyces cerevisiae is initiated by the formation of DNA double strand breaks (DSBs), which are created by Spoil protein. Recombination preferentially occurs between homologous chromosomes, in order to establish interhomologue connections. These connections serve as a platform for genetic recombination and to promote accurate homologue disjunction at the first meiotic division (MI). Specific mechanisms are in place to ensure that meiotic DSB repair is directed towards interchromosomal repair, and genes thought to be involved in these mechanisms were examined in a DSB assay, where interchromosomal repair was precluded. Genes involved in the formation and processing of Spol1-DSBs were also examined. In meiosis, the regulation of resectioning is critical to repair outcome, and this assay was designed to measure two different lengths of resection tract. In a mek1 mutant, there was an increase in the generation of longer resection tracts, suggesting that Mek1 protein may exert its influence over repair template choice by negatively regulating DSB resectioning. An sae2 mutant was found to generate fewer shorter resection tracts, and was delayed for DSB repair. This suggested that Sae2 protein may have an early role in resectioning, by influencing repair template choice. Mutants of the MRX complex were all compromised for DSB repair, while an exol mutant failed to generate long resection tracts only. Finally, from work on a dmcl mutant, the prospect of protein sequestration at sites of excess single stranded DNA was proposed.