Title

Regulation of DNA replication and repair by DNA polymerase delta interacting protein 1 (PDIP1)

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

The core enzyme of DNA polymerase delta (pol delta), an essential DNA polymerase for chromosomal DNA replication in eukaryotes, is comprised of a large catalytic subunit (p125) and a small subunit (p50) whose function is unclear. Identification of the direct interaction of p50 with the Proliferating Cell Nuclear Antigen (PCNA), the processivity factor of pol delta, as well as its essential role in mediating the stimulatory effect of PCNA on pol delta, suggested that p50 might be a potential organizer that mediates the regulatory effects of protein factors on pol delta. As an approach to understand the function of the small subunit of pol delta, a novel protein, Polymerase Delta Interacting Protein 1 (PDIP1), was previously identified from a yeast two-hybrid screen of a HepG2 cDNA library using p50 as bait (He et al., 2001). In that study, PDIP1 was also found to interact with PCNA and a putative PCNA binding motif was identified in the C-terminus of PDIP1. In the present study, a second PCNA binding site was identified in the N-terminal region of PDIP1. The interaction of PDIP1 with PCNA was demonstrated to be mediated through these two PCNA binding sites which were found to have different binding affinities for PCNA under specific conditions. Indirect immunofluorescence and confocal microscopy studies showed that PDIP1 colocalizes with PCNA at distinct nuclear foci during S phase and following UV irradiation, suggesting that PDIP1 may be involved in both DNA replication and DNA repair. Furthermore, it was found that the C-terminal PCNA binding site is essential for the colocalization of PDIP1 with PCNA following UV irradiation, whereas the N-terminal PCNA binding site is more important for the colocalization during S phase, suggesting that each PCNA binding site may play a different role in DNA repair or DNA replication. Flow cytometry studies showed that overexpressing PDIP1 in HeLa cells accelerated S phase entry and that the N-terminal PCNA binding site may be responsible for this stimulation, suggesting that PDIP1 may have an important role in regulating DNA synthesis through its interaction with PCNA at the N-terminal PCNA binding site.

Keywords

Biology, Molecular

Link to Full Text

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