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

Abstract

ABSTRACTDNA polymerase delta (Polδ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Polδis responsible for the synthesis and processing of the lagging-strand. At replication origins, Polδhas been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Polδsubunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of PolδinSaccharomyces cerevisiaelead to pervasive genome instability. Here, we analyze how the depletion of Polδimpacts replication origin firing and lagging-strand synthesis during replication elongationin vivoinS. 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 Polδdepletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Polδis capable of synthesizing a substantial amount of lagging-strand DNA, even when Polδis severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Polδis 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 (Polδ) is a multi-functional replisome enzyme responsible for synthesis and processing of the lagging-strand. Mutations in Polδcause a variety of human diseases: for example, destabilizing mutations lead to immunodeficiency. We titrate the concentration of Polδin budding yeast – a simple model eukaryote with conserved DNA replication machinery. We characterize several replication defects associated with Polδscarcity. The defects we observe provide insight into how destabilizing Polδmutations lead to genome instability.