Efficient repair of large DNA loops in Saccharomyces cerevisiae.

Small looped mispairs are efficiently corrected by mismatch repair. The situation with larger loops is less clear. Repair activity on large loops has been reported as anywhere from very low to quite efficient. There is also uncertainty about how many loop repair activities exist and whether any are conserved. To help address these issues, we studied large loop repair in Saccharomyces cerevisiae using in vivo and in vitro assays. Transformation of heteroduplexes containing 1, 16 or 38 nt loops led to >90% repair for all three substrates. Repair of the 38 base loop occurred independently of mutations in key genes for mismatch repair (MR) and nucleotide excision repair (NER), unlike other reported loop repair functions in yeast. Correction of the 16 base loop was mostly independent of MR, indicating that large loop repair predominates for this size heterology. Similarities between mammalian and yeast large loop repair were suggested by the inhibitory effects of loop secondary structure and by the role of defined nicks on the relative proportions of loop removal and loop retention products. These observations indicate a robust large loop repair pathway in yeast, distinct from MR and NER, and conserved in mammals.

Correction of large mispaired DNA loops by extracts of Saccharomyces cerevisiae.

Single base mispairs and small loops are corrected by DNA mismatch repair, but little is known about the correction of large loops. In this paper, large loop repair was examined in nuclear extracts of yeast. Biochemical assays showed that repair activity occurred on loops of 16, 27, and 216 bases, whereas a G-T mispair and an 8-base loop were poorly corrected under these conditions. Two modes of loop repair were revealed by comparison of heteroduplexes that contained a site-specific nick or were covalently closed. A nick-stimulated repair mode directs correction to the discontinuous strand, regardless of which strand contains the loop. An alternative mode is nick-independent and preferentially removes the loop. Both outcomes of repair were largely eliminated when DNA replication was inhibited, suggesting a requirement for repair synthesis. Excision tracts of 100-200 nucleotides, spanning the position of the loop, were observed on each strand under conditions of limited DNA repair synthesis. Both repair modes were independent of the mismatch correction genes MSH2, MSH3, MLH1, and PMS1, as judged by activity in mutant extracts. Together the loop specificity and mutant results furnish evidence for a large loop repair pathway in yeast that is distinct from mismatch repair.