Reduced cellular levels of DNA polymerase delta alter replication-fork dynamics and enzymology, and globally impair lagging-strand processing

DNA polymerase delta (Pol ∂) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol ∂ is responsible for the synthesis and processing of the lagging strand; this role requires Pol ∂ to extend Okazaki fragment primers synthesized by Pol ⍺-primase, and to carry out strand-displacement synthesis coupled to nuclease cleavage during Okazaki fragment termination. Destabilizing mutations in human Pol ∂ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol ∂ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze the how the depletion of Pol ∂ impacts replication initiation and elongation in vivo in S. cerevisiae. We determine that Pol ∂ depletion leads to a dependence on checkpoint signaling and recombination-mediated repair for cellular viability. By analyzing nascent lagging-strand products, we observe both a genome-wide change in the establishment and progression of replication forks and a global defect in Pol ∂-mediated Okazaki fragment processing. Additionally, we detect significant lagging-strand synthesis by the leading-strand polymerase (Pol ɛ) in late regions of the genome when Pol ∂ is depleted. Pol3 levels were altered by treatment with IAA in a strain containing an auxin-inducible degron on Pol3 and the resulting Okazaki fragments were sequenced from asynchronous and synchronous cultures. Addtionally, changes in ribonucleotide incorporation by ribonucleotide-hyperincorporating strains was assesed by HydEN-seq in order to determine the changes in polymerase usage during Pol3 depletion.