The Rrm3p DNA helicase promotes genome integrity

Rrm3p is a 5′ to 3′ DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the S. cerevisiae genome. Stalled and broken replication forks incurred in rrm3 cells activate the intra-s phase checkpoint that functions to slow down the cell cycle in order to repair DNA damage. This thesis focuses on the role of Rrm3p in replication fork progression past local chromatin structures and the cellular responses that are necessary to repair DNA damage incurred in rrm3 cells.; To analyze Rrm3p's role in replication past protein-DNA complexes a cis acting site within the ribosomal DNA (rDNA) locus called the replication fork barrier (RFB) was analyzed. Fob1p bound the RFB throughout the cell cycle, and this binding was required for the rrm3-generated defects in fork progression at the RFB. None of the rrm3-generated rDNA replication defects were reduced by lack of the Sir2p histone deacetylase, which affects chromatin structure throughout the rDNA. These data suggest that local chromatin structure makes replication dependent upon Rrm3p.; To understand the mechanisms that respond to and repair rrm3 -dependent lesions, we identified genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S phase and DNA damage checkpoints, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, the MRE11/RAD50/XRS2 (MRX) nuclease and break induced replication (BIR) were all important for viability or normal growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.