Opportunities for new studies of nuclear DNA replication enzymology in budding yeast.

Three major eukaryotic DNA polymerases, Polymerases α, δ, and ε (Pols α, δ, and ε), perform the fundamental process of DNA synthesis at the replication fork both accurately and efficiently. In trying to understand the necessity and flexibility of the polymerase usage, we recently reported that budding yeast cells lacking Pol ε exonuclease and polymerase domains (pol2-16) survive, but have severe growth defects, checkpoint activation, increased level of dNTP pools as well as significant increase in the mutation rates. Herein, we suggest new opportunities to distinguish the roles of Pol ε from those of two other eukaryotic B-family DNA polymerases, Pols δ and ζ.

Roles for DNA polymerase δ in initiating and terminating leading strand DNA replication.

Most current evidence indicates that DNA polymerases ε and δ, respectively, perform the bulk of leading and lagging strand replication of the eukaryotic nuclear genome. Given that ribonucleotide and mismatch incorporation rates by these replicases influence somatic and germline patterns of variation, it is important to understand the details and exceptions to this overall division of labor. Using an improved method to map where these replicases incorporate ribonucleotides during replication, here we present evidence that DNA polymerase δ universally participates in initiating leading strand synthesis and that nascent leading strand synthesis switches from Pol ε to Pol δ during replication termination. Ribonucleotide maps from both the budding and fission yeast reveal conservation of these processes. These observations of replisome dynamics provide important insight into the mechanisms of eukaryotic replication and genome maintenance.

The absence of the catalytic domains of Saccharomyces cerevisiae DNA polymerase ϵ strongly reduces DNA replication fidelity.

The four B-family DNA polymerases α, δ, ϵ and ζ cooperate to accurately replicate the eukaryotic nuclear genome. Here, we report that a Saccharomyces cerevisiae strain encoding the pol2-16 mutation that lacks Pol ϵ's polymerase and exonuclease activities has increased dNTP concentrations and an increased mutation rate at the CAN1 locus compared to wild type yeast. About half of this mutagenesis disappears upon deleting the REV3 gene encoding the catalytic subunit of Pol ζ. The remaining, still strong, mutator phenotype is synergistically elevated in an msh6Δ strain and has a mutation spectrum characteristic of mistakes made by Pol δ. The results support a model wherein slow-moving replication forks caused by the lack of Pol ϵ's catalytic domains result in greater involvement of mutagenic DNA synthesis by Pol ζ as well as diminished proofreading by Pol δ during replication.

Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae.

To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication. Ribonucleotides are incorporated into the pol2-16 genome in patterns consistent with leading-strand replication by Pol δ when Pol ε is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.