Role of yeast DNA polymerase epsilon during DNA replication

Sammanfattning: Each cell division, the nuclear DNA must be replicated efficiently and with high accuracy to avoid mutations which can have an effect on cell function. There are three replicative DNA polymerases essential for the synthesis of DNA during replication in eukaryotic cells. DNA polymerase ? (Pol ?) synthesize short primers required for DNA polymerase ? (Pol ?) and DNA polymerase ? (Pol ?) to carry out the bulk synthesis. The role of Pol ? and Pol ? at the replication fork has been unclear. The aim of this thesis was to examine what role Pol ? has at the replication fork, compare the biochemical properties of Pol ? and Pol ?, and to study the function of the second largest and essential subunit of Pol ?, Dpb2.To identify where Pol ? replicates DNA in vivo, a strategy was taken where the active site of Pol ? was altered to create a mutator polymerase leaving a unique error-signature. A series of mutant pol ? proteins were purified and analyzed for enzyme activity and fidelity of DNA synthesis. Two mutants, M644F and M644G, exhibited an increased mutation rate and close to normal polymerase activity. One of these, the M644G gave rise to a specific increase of mismatch mutations resulting from T-dTMP mis-pairing during DNA synthesis in vitro. The M644G mutant was introduced in yeast strains carrying a reporter gene, URA3, on either side of an origin in different orientations. Mutations which inactivated the URA3 gene in the M644G mutant strains were analyzed. A strand specific signature was found demonstrating that Pol ? participates in the synthesis of the leading strand.Pol ? and Pol ? are both stimulated by the processivity clamp, PCNA, in in vitro replication assays. To clarify any differences they were challenged side by side in biochemical assays. Pol ? was found to require that single-stranded template (ssDNA) was entirely coated with RPA, whereas Pol ? was much less sensitive to uncoated ssDNA. The processivity of Pol ? was stimulated to a much higher degree by PCNA than of Pol ?. In presence of PCNA the processivity of Pol ? and Pol ? was comparable. In contrast, Pol ? was approximately four times slower than Pol ? when replicating a single-primed circular template in the presence of all accessory proteins and an excess of polymerase. The biochemical characterization of the system suggests that Pol ? and Pol ? are loaded onto the PCNA-primer-ternary complex by separate mechanisms. A model is proposed where the loading of Pol ? onto the leading strand is independent of the PCNA interaction motif which is required by enzymes acting on the lagging strand.The essential gene DPB2 encodes for the second largest subunit of Pol ?. We carried out a genetic screen in S.cerevisiae and isolated a lethal mutant allele of dpb2 (dpb2-200). When over-expressed together with the remaining three subunits of Pol?, Pol2, Dpb3 and Dpb4, the dpb2-201 did not copurify. The biochemical property of Pol2/Dpb3/Dpb4 complex was compared with wild-type four-subunit Pol ? (Pol2/Dpb2/Dpb3/Dpb4) and a Pol2/Dpb2 complex in replication assays. The absence of Dpb2 in the complex did not significantly affect the specific activity or the processivity, but gave a slightly reduced efficiency in holoenzyme assays when compared to wild-type four-subunit Pol ?. We propose that Dpb2 is not essential for the enzyme activity of Pol ?.