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 ∂) 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-replicating regions of the genome when Pol ∂ is depleted. Together, our results are consistent with the presence of least two Pol ∂ complexes in the eukaryotic replisome under normal conditions. We propose that replisomes with sub-stoichiometric Pol ∂ are defective due to competition between nascent lagging-strand primers and terminating Okazaki fragments, and that this competition underlies both the replication and genome stability defects observed when cellular levels of Pol ∂ fall below a critical threshold. Supplementary figure 1 (associated with figure 1). LEGENDS TO SUPPLEMENTARY FIGURES Further characterization of Pol3 depletion kinetics and the effect of depletion on growth rate.A. Western blot against Pol3-9Myc or TIR1-9Myc in asynchronous cultures of the POL3-AID strain following 30 minutes of treatment with the indicated concentration of IAA B. Growth rates of POL3-AID cells in liquid culture. Data were calculated from three replicates.C. DNA content measured by flow cytometry for logarithmically growing POL3-AID cells treated with the indicated concentration of IAA for 2hSupplementary figure 2 (associated with figure 3). Effects of Pol3 depletion on replication origin firing in individual replicates.A-F. Analysis of replication origin firing efficiency as shown for pooled replicates in Fig. 3B,D&E, separated by individual biological replicate strain as indicated. **** denotes p < 0.0001 by Kolmogorov-Smirnov test (A,D) or Wilcoxon signed rank test (B-C, E-F) for a change in the distribution or mean of the data, respectively. Supplementary figure 3 (associated with figure 4) Samples used for analysis of replication-fork speed in figure 4.DNA content measured by flow cytometry for the samples shown in Fig. 4A. Red timepoints were sequenced for both 0 and 1 mM IAA, and blue timepoints for 1 mM only. figure 4 (associated with figure 5) Addi...