PT  - JOURNAL ARTICLE
AU  - Natasha C Koussa
AU  - Duncan J. Smith
TI  - Reduced cellular levels of DNA polymerase delta alter replication dynamics and enzymology, and impair lagging-strand processing
AID  - 10.1101/2019.12.17.879544
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 2019.12.17.879544
4099  - http://biorxiv.org/content/early/2019/12/18/2019.12.17.879544.short
4100  - http://biorxiv.org/content/early/2019/12/18/2019.12.17.879544.full
AB  - 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 α-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.