Accumulation of single-stranded DNA and destabilization of telomeric repeats in yeast mutant strains carrying a deletion of RAD27

The Saccharomyces cerevisiae RAD27 gene encodes the yeast homologue of the mammalian FEN-1 nuclease, a protein that is thought to be involved in the p rocessing of Okazaki fragments during DNA lagging-strand synthesis. One of the predicted DNA lesions occurring in rad27 strains is the presence of sin gle-stranded DNA of the template strand for lagging-strand synthesis. We ex amined this prediction by analyzing the terminal DNA structures generated d uring telomere replication in rad27 strains. The lengths of the telomeric r epeat tracts were found to be destabilized in rad27 strains, indicating tha t naturally occurring direct repeats are subject to tract expansions and co ntractions in such strains. Furthermore, abnormally high levels of single-s tranded DNA of the templating strand for lagging-strand synthesis were obse rved in rad27 cells. Overexpression of Dna2p in wild-type cells also yielde d single-stranded DNA regions on telomeric DNA and caused a cell growth arr est phenotype virtually identical to that seen for rad27 cells grown at the restrictive temperature. Furthermore, overexpression of the yeast exonucle ase Exo1p alleviated the growth arrest induced by both conditions, overexpr ession of Dna2p and incubation of rad27 cells at 37 degrees C. However, the telomere heterogeneity and the appearance of single-stranded DNA are not p revented by the overexpression of Exo1p in these strains, suggesting that t his nuclease is not simply redundant with Rad27p, Our data thus provide in vivo evidence for the types of DNA lesions predicted to occur when lagging- strand synthesis is deficient and suggest that Dna2p and Rad27p collaborate in the processing of Okazaki fragments.