Title

Identification and characterization of a novel protein that interacts with the 50-kDa subunit of human DNA polymerase delta and PCNA

Date of Award

2000

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Antero G. So, Committee Chair

Abstract

DNA polymerase 8 which plays primary roles in DNA replication and repair in eukaryotic cells, is comprised of at least two essential subunits of 125- and 50-kDA. The large catalytic subunit possesses both DNA polymerase and 3' to 5' exonuclease activities whereas the function of the small subunit until now is not known. As an approach to understand the function of the 50-kDa subunit (p50), the yeast two-hybrid system was used to screen a HepG2 library for proteins that interact with this subunit. A cDNA encoding a novel protein of 36-kDa, polymerase delta interacting protein 1 (PDIP1), that specifically interacts with 50-kDA subunit but not the 125-kDa, was identified. The interaction of p50 and PDIP1 was further confirmed by a pull-down assay using a GST-PDIP1 fusion protein. PDIP1 was further demonstrated to interact with the proliferating cell nuclear antigen (PCNA) using the same binding assay. Sequence comparisons demonstrate that PDIP1 is distinct from the postulated third subunit of DNA polymerase delta (fission yeast cdc27, budding yeast POL32 and mammalian p66). A putative PCNA binding motif was identified within the C-terminus of PDIPI. A synthetic peptide containing this PCNA binding motif was shown to bind to PCNA CNA by Far Western analysis, but not a synthetic peptide in which two critical amino acids were substituted (V252A and F254A). Immunofluoresence staining experiments demonstrated that PDIP1 co-localized with p50, PCNA and BrdU at replication foci. Taken together this suggests that PDIP1 is involved in DNA replication/repair, and that PDIP1 may modulate the interaction of PCNA with the 50-kDa subunit of pol delta. Consistent with this suggestion is the observation that the stimulation of pol delta activity by PDIP1 is dependent on the presence of PCNA.

Keywords

Biology, Molecular

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9992511