David J. Phillips

ASSESSING THE ROLES OF A NOVEL DOMAIN WITHIN THE YEAST PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) DURING DNA REPLICATION AND REPAIR. David J. Phillips¹, Bonita L. Yoder², Adam Wood², Peter M.J. Burgers²; Department of Biology, Washington University, St. Louis, MO¹; Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO²

The eukaryotic replication clamp proliferating cell nuclear antigen (PCNA) is an essential processivity factor and regulatory switch in multiple metabolic pathways, including DNA replication, damage avoidance, and cell cycle control. PCNA is a homotrimer of 29-kDa subunits whose toroidal structure and hollow central cavity allow for favorable interaction with double-stranded DNA. Once loaded onto DNA by Replication Factor-C (RFC), PCNA recruits the appropriate molecular machinery to the replication fork given the cellular environment and possible DNA damage encountered. The Burgers laboratory has been mapping the domains of the Saccharomyces cerevisiae PCNA through a series of deletion analyses and has elucidated the pathways associated with a number of interaction sites. The anterior face of PCNA faces the direction of DNA replication and contains the majority of conserved protein interaction domains essential to cellular survival and known DNA metabolic processes, including sites of ubiquitination and binding of both replicatory and translesion DNA polymerases.

Recently, the laboratory developed interest in a previously unexamined conserved domain on the posterior face of PCNA, a rocket-like projection of nine amino acids with a solvent-exposed hydrophobic Methionine residue at the apex, which we hypothesize may be involved in protein-protein interactions with an as yet determined factor. In order to determine the importance of this domain, a mutant was created, pcna-95, wherein the POL30 gene encoding the protein was altered to remove the nine amino acids comprising the loop and replace them with a GGAG motif, creating a sharp turn with minimal projection. A plasmid containing the mutant gene was inserted into appropriate yeast strains for in vivo genetic assays and into E. coli for over expression of the protein for use in in vitro biochemical assays.

The major focus of this project was to determine which DNA damage response pathways, if any, pcna-95 is defective for, thus elucidating the importance of the rocket-like posterior projections. Compared to wild type PCNA, pcna-95 in vitro demonstrates comparable rates of lagging-strand DNA replication by DNA polymerase delta and translesion synthesis by polymerase eta and Rev1. In vivo, strains transformed with the mutant gene pol30-95 respond similarly to those containing the wild type PCNA gene during assays of spontaneous mutagenesis and cytotoxic response to the interstrand cross-linking agent Nitrogen Mustard. However, strains of yeast carrying the pol30-95 mutant display slightly increased sensitivity to UV damage and slightly decreased rates of UV damage-induced mutagenesis when compared to wild type strains, suggesting a possible association and/or cooperation of our novel domain with the molecular machinery responsible for DNA damage repair.

As this examination of the mutant pcna-95 moves forward, we plan to study the possible involvement of the posterior hydrophobic projections in alternate metabolic pathways, particularly, those associated with regulation of chromatin silencing.