Title

Studies on the roles of DNA polymerases delta and epsilon in DNA replication and repair, and the interaction of the small subunit of DNA polymerase delta with proliferating cell nuclear antigen

Date of Award

2006

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Antero G. So, Committee Chair

Abstract

At least three DNA polymerases (alpha, delta and epsilon) are required for normal chromosomal DNA replication in eukaryotes. Pol alpha has been shown to be responsible for the synthesis of RNA-DNA primers to initiate Okazaki fragment synthesis. However, the precise role of either pol delta or pol epsilon until now has not been elucidated. Pol delta but not pol epsilon has been found to be the DNA polymerase activity required to complete the replication of SV40 genome in vitro (Waga and Stillman, 1998). Previous studies in both budding and fission yeast indicated that the N-terminal portion of the catalytic subunit of pol epsilon containing the polymerase and exonuclease activities are not essential for cell viability (Kesti et al., 1999; Feng and D'Urso, 2001), although cells lacking this catalytic domain show some S-phase defects, and the point mutation inside the catalytic site is lethal (Dua et al., 1999). In this study we have investigated the respective roles of pol delta and pol epsilon in DNA replication by using indirect immunofluorescence and confocal microscopy to analyze the patterns of co-localization of each DNA polymerase with PCNA at replication foci during the S-phase of the cell cycle. Our study demonstrated that the catalytic subunit of pol delta co-localizes with PCNA throughout S-phase whereas the catalytic subunit of pol epsilon co-localizes with PCNA mainly in late S-phase. These two polymerases also co-localize with PCNA after UV irradiation, however, the dosage of UV or the recovery time required for the co-localization is different for pol delta and pol epsilon.Despite the fact that proliferating cell nuclear antigen (PCNA) was first identified as a processivity factor for pol delta about 20 years ago (Tan et al., 1986; Prelich et al., 1987), the identity of the subunit of pol delta that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme remains controversial. All three major subunits of pol delta (p125, p50 and p66) have been reported to contain a PCNA-binding motif and the interaction of each subunit with PCNA has been suggested to be primarily responsible for the high processivity of pol delta. We have recently demonstrated a direct interaction between human recombinant p50 and PCNA by reciprocal co-immunoprecipitation of the two proteins using antibodies against either p50 or PCNA (Lu et al., 2002) and have further identified a PCNA-binding motif (MRPFL) at the N terminus of p50. In the present study, experiments were designed to determine whether the identified PCNA-binding motif of p50 is essential for its interaction with PCNA as well as for its localization with PCNA at replication and repair foci during active DNA synthesis in living cells. In these studies we have demonstrated that substitution and deletion mutants of p50 were defective in interaction with PCNA, as measured by co-immunoprecipitation of transfected c-Myc tagged p50 with HA tagged PCNA in HeLa cell lysates. Furthermore, co-localization of p50 and PCNA at replication and repair foci was reduced or eliminated by mutation of the PCNA-binding motif. These findings establish that the PCNA-binding motif in p50 is functionally required for the interaction of pol delta with PCNA.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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