Molecular mechanism of mating-type switching in Schizosaccharomyces pombe

pombe的单倍体种群包含两种不同细胞类型的比例：P和m.s. pombe已经开发出一种称为交配类型转换（MTS）的优雅机制，允许它改变交配类型/细胞类型。当两个基因座之间的遗传信息通过同源重组（HR）依赖机制进行交换时，就会发生MTS。HR是一种DNA修复机制，可以使用高度相似的完整DNA片段修复双链断裂。通过对MTS缺陷基因的全面遗传筛选，鉴定出两个高度保守的基因为新的致病因子。其中一个分离的候选因子作为辅助因子刺激Rad51重组酶。Rad55-Rad57配合物已成功纯化到接近均匀性，但生物化学表征已被证明是困难的。另外，我们利用Rad51 - i308a、I308T和E206A点突变体来了解Rad55-Rad57对Rad51介导的HR的影响。点突变体在Rad55-Rad57通路上存在缺陷，但这种缺陷不是由于体内复合物形成的破坏。每个突变体与Rad55-Rad57的直接物理相互作用也与野生型Rad51相当。通过常规的链交换实验，我证明I308A和I308T点突变体的活性比E206A和野生型低2倍。I308A似乎形成了更稳定的灯丝，I308T和E206A形成的灯丝表现出与野生型Rad51相似的稳定性。我还看到每个突变体在ssDNA上的关联与野生型相似。

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.