Unraveling differential origin firing and replication stress mediated genome instability using S. cerevisiae as a model organism

Abstract

Yeasts have been used as model organisms because of their compact genome size, simple growth requirements, and homology to higher eukaryotes. The sites for origin initiation are well defined in the S. cerevisiae genome, and several metabolism and resistance genes are well conserved to higher eukaryotes, making it an ideal model system to study origin dynamics and replication inhibitor mediated genome instability. In this thesis, using S. cerevisiae as a model we answered two big questions. First, we studied factors regulating differential origin firing between two commonly used laboratory strains. Second, we identified the general mechanism used by replication inhibitors to induce genome-wide DSBs. To identify the factors regulating differential origin firing we compared, 1) S-phase progression, 2) the relative level of checkpoint protein, and 3) the binding of Rad53 and Cdc45 at a select few origins. Amongst the three, we found that difference in binding of Rad53 at the origins was responsible for differential firing between the two strains. In a new origin class, "Rad53 dependent" we looked the role of active transcription in regulating origin firing. Our results show that active transcription during S-phase does not wipe out the origin firing activity. To study replication inhibitor-induced genome instability, we used a broad range of inhibitors that induce replication stress differently. Our results suggested that replication-transcription collision is a common mechanism used by these inhibitors to induce DSBs. Associating each DSB with an upregulated gene, we found an enrichment in the head-on orientations. As these inhibitors are regularly used in the clinic as anticancer therapeutics, studying the gene expression helped us identify their mechanism of action to specifically target cancer cells. We found that all of these individually upregulate the oxidation-reduction pathway, and downregulate glycolysis. Finally, at one locus on chromosome II which induces DSB specifically in CPT, we were able to demonstrate replication fork pause and correlation of the DSB with active transcription-deposited histone marks.