The Function Of Checkpoint Kinase Rad3and Rad17in Homologous Recombination Repair

A key function of the replication checkpoint is to prevent replication forks catastrophically failing. To achieve this, the S. pombe checkpoint kinases Rad3and Cdsl regulate proteins that affect DNA metabolism, including Exol, Mus81and Rad60. Here we use a site-specific replication fork arrest system to distinguish between these known roles at stable stalled forks from their potential functions at a collapsed fork. We reveal antagonistic roles for the core checkpoint kinase Rad3and the9-1-1clamp:Rad3restrains recombination protein-association while the9-1-1complex promotes association at collapsed forks. We further demonstrate that Rad3restrains, and9-1-1promotes, recombination and genome stability behind a collapsed fork.By chromatin immunoprecipitation (ChIP) we previously demonstrated that, upon the induction of replication fork arrest at RTSl, Rad22is specifically recruited to the uraR locus. In the wild-type strain background,"arrest on" conditions in the rad3.d strains resulted in Rad22, RPA, Rhp51recruitment at and immediately flanking the uraR locus. In contrast, in the radl7.d strains, proteins recruitment was significantly reduced at RTS1sequences and at both the telomere and centromere proximal flanking sequences relative to wild-type. In the exo1.d strains, Rad22, Rhp51recuitment was reduced at RTS1, for RPA protein had no obvious impact.In wild-type cells, the induction of fork arrest stimulated ade6recombination6.9-fold over the baseline frequency In the rad3.d mutant, recombination was stimulated12.5-fold over baseline with replication pause "on". In the radl7.d mutant, recombination was stimulated by just3.7-fold. Taken together, these data suggest that RTS1-induced ade6heteroallele recombination is suppressed by Rad3and promoted by Rad17. It is likely that Rad3exerts its effects in the ade6system by suppressing RPA and Rad22recruitment behind RTS1, and Rad17exerts its effect via promoting recruitment.