Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair

Abstract

DNA polymerase delta (Pol{delta} ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol{delta} is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol{delta} has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol{delta} subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol{delta} in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol{delta} impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol{delta} depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol{delta} is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol{delta} is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol{delta} is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability. SIGNIFICANCE STATEMENTDNA replication in eukaryotes is carried out by the replisome - a multi-subunit complex comprising the enzymatic activities required to generate two intact daughter DNA strands. DNA polymerase delta (Pol{delta} ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Pol{delta} cause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Pol{delta} in budding yeast - a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Pol{delta} scarcity. The defects we observe provide insight into how destabilizing Pol{delta} mutations lead to genome instability.