Molecular mechanism of mating-type switching in Schizosaccharomyces pombe

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A haploid population of S. pombe contains a proportional number of two different cell types: P and M. S. pombe has developed an elegant mechanism called mating type switching (MTS) that allows it to change mating type/cell type. MTS occurs when the genetic information between two loci are exchanged through a homologous recombination (HR)-dependent mechanism. HR is a DNA repair mechanism that can repair a double-strand break using a highly similar segment of intact DNA. Through a comprehensive genetic screening of genes defective in MTS, two highly conserved genes were identified as novel factors. One of the isolated candidates functions as an auxiliary factor to stimulate the Rad51 recombinase.Rad55-Rad57 complex has been successfully purified to near homogeneity however biochemical characterisation has proven difficult to tract. Alternatively rad51-I308A, I308T and E206A point mutants are utilised to understand Rad55-Rad57 effect on Rad51 mediated HR. The point mutants are defective in the Rad55-Rad57 pathway, however this defect is not due disruption of in vivo complex formation. Direct physical interaction of each mutant with Rad55-Rad57 is also comparable to wild type Rad51. Using conventional strand exchange assay I demonstrated that I308A and I308T point mutants are 2 fold less active compared to E206A and wildtype. I308A seemed to form a more stable filament and filaments formed by I308T and E206A exhibited similar stability to wild type Rad51. I also saw that the association of each mutant on ssDNA was similar to wildtype.