Mutator phenotype induced by aberrant replication

We have identified thermosensitive mutants of five Schizosaccharomyces pomb e replication proteins that have a mutator phenotype at their semipermissiv e temperatures, Allele-specific mutants of DNA polymerase delta (pol delta) and mutants of Pol alpha, two Pol delta subunits, and ligase exhibited inc reased rates of deletion of sequences flanked by short direct repeats. Dele tion of rad2(+), which encodes a nuclease involved in processing Okazaki fr agments, caused an increased rate of duplication of sequences flanked by sh ort direct repeats, The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2 Delta background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2 Delta nas also reduced in a thermosensitive polymerase background, bu t not in a ligase mutator background, which suggests that formation of dupl ication mutations requires normal DNA polymerization. Thus, although the de letion and duplication mutator phenotypes are distinct, their mutational me chanisms are interdependent, The deletion and duplication replication mutat ors all exhibited decreased viability in combination with deletion of a che ckpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kina se functioning in a checkpoint Rad mediated reversible S-phase arrest pathw ay, decreased the viability and exacerbated the mutation rate only in the t hermosensitive deletion replication mutators but had no effect on rad2 Delt a. These findings suggest that aberrant replication caused by allele-specif ic mutations of these replication proteins can accumulate potentially mutag enic DNA structures. The checkpoint Rad-mediated pathways monitor and signa l the aberrant replication in both the deletion and duplication mutators, w hile Cds1 mediates recovery from aberrant replication and prevents formatio n of deletion mutations specifically in the thermosensitive deletion replic ation mutators.