Analysis of replication barriers in helicase mutants of the model eukaryote Saccharomyces cerevisiae

Sequences that assume non B-DNA structures and sequences that are targets of protein complexes are potent sources of DNA instability because of their potential to interfere with the process of replication. Accordingly, important roles have been attributed to DNA helicases in preserving DNA stability by assisting normal replication through sequence and protein barriers. In order to understand how eukaryotic cells replicate across sequence and protein barriers, the progression of DNA replication across sequence and protein barriers was analyzed in vivo in the model eukaryote Saccharomyces cerevisiae. Replication of hairpin-forming CAG/CTG and CGG/CCG repeat sequences, expansions of which are implicated in several human diseases, G-quadruplex forming Oxytricha and human telomeric sequences, and yeast telomeric sequences that are targets of Rap1 protein complexes were analyzed in helicase mutants that included srs2Delta, sgs1Delta, pif1Delta and rrm3Delta. Two-dimensional gel analysis of replication of CTG repeat containing locus showed an altered pattern of replication with abundant joint molecules (JMs), which migrated like reversed replication forks, and whose presence was dependent on SRS2 and SGS1, but not RAD51 . These results suggested that Srs2 promoted fork reversal in repetitive sequences. Analysis of sequence changes of the hairpin-forming CAG/CTG repeats showed that CAG/CTG repeats underwent more frequent expansions, contractions and breakage in the absence of Srs2 helicase. These changes were dependent on the DNA strand exchange factor Rad51, suggesting that unrestrained recombination triggers trinucleotide repeat instability in Srs2 deficient cells. In contrast, in the absence of the Sgs1 helicase, CAG/CTG repeats underwent frequent contractions and breakage, an effect that was partially dependent on Rad51 or Rad52. Using various domain mutants of the Srs2 helicase, we found that the contractions and expansions phenotypes of CAG/CTG repeats can be uncoupled from their breakage phenotype. The Srs2 helicase was indentified as having an important role in assisting replication through hairpin forming CGG/CCG repeats, preventing their fragility. Rrm3 helicase was identified as an important factor involved in the replication of the protein-mediated yeast telomeric barrier and preventing recombination. The Sgs1 and Pif1 helicases were found to have no major roles in unwinding G-quadruplex structures in vivo. The experiments with hydroxyurea indicated that Tof1 dependent and independent mechanisms maintain protein-mediated stalls. Finally, a novel stall site was identified at the 3' end of URA3 gene on the CF1 YAC that was partially dependent on Tof1 and not due to any identifiable structure-forming sequences, therefore suggesting the likely collision of the replication-fork with a protein-complex. In summary, this thesis research has identified key aspects of replication through sequence and protein barriers.