The limiting DNA replication initiation factors Sld2 and Sld3 influence replication origin efficiency independent of time of origin firing in Saccharomyces cerevisiae

Abstract

During S-phase in Saccharomyces cerevisiae, DNA replication is initiated from roughly 300 chromosomal origins that are regulated both by DNA sequence and by interacting protein factors. The limited abundance of a set of trans-acting initiation proteins—Sld2, Sld3, Dpb11 and Dbf4, collectively called “SSDD”— gives rise to the disparity in origin activation observed across the genome. The biological significance of this variable firing time and efficiency—conserved features among eukaryotes—is not well understood, nor is it clear how SSDD limitation leads to differences in origin firing time and efficiency. I wanted to determine whether the association of individual SSDD factors with the assembling replication complex contributes to both time of origin activation and origin efficiency. I used auxin-induced protein degradation to further exacerbate the SSDD limitation, focusing on the consequences for genome duplication when the abundance of either Sld2 or Sld3 is reduced. I found that depleting cells of either initiation factor slows growth rate, increases S-phase duration, and causes viability defects, none of which are due to activation of the S phase checkpoint. Using genome-wide replication assays, I found that, compared to wild type, the same set of origins is active even when Sld2 or Sld3 are depleted from cells, implying that time of origin firing does not change in response to the availability of these two factors. Instead, I discovered that replication forks move farther when either Sld2 or Sld3 is depleted, indicating that origin firing efficiency is reduced under both conditions. I confirmed these changes to origin efficiency by 2-dimensional gel electrophoresis of multiple individual origins. The viability defects induced by the depletion of these two limiting initiation factors suggested that the genome is destabilized by the systemic reduction of replication efficiency. Using pulsed-field gel electrophoresis to assay the integrity of whole chromosomes over the course of synchronized Sld2- or Sld3-depleted S phase, I found that all chromosomes except ChrXII are stable. Breakage occurs within the rDNA locus on ChrXII, likely due to its incomplete replication, which is not surveilled by the S phase checkpoint. In summary, the cellular abundance of Sld2 and Sld3 contributes mostly to origin firing efficiency. In contrast to a recently proposed idea that origin firing time is a secondary consequence of origin efficiency, my work detailed in this dissertation supports the concept that efficiency and time of initiation are separable features of the eukaryotic genome.