Using an immunoaffinity column procedure, Dr. Lee and her coworkers have achieved rapid purification of significant amounts of polymerase delta in a 3 subunit form, i.e. p125, p50 and a new p75 subunit or a tightly associated accessory protein. The p125 gene has been cloned and expressed in E. coli, mammalian cells and insect cells that yield large amounts of post transcriptionally processed and modified (in mammalian and insect cells) and nonmodified (in E. coli) enzyme. The p50 and p75 subunits will also be cloned and expressed and interaction of the various subunits and its effect on the properties of the core enzyme will be studied by a variety of standard procedures. The single p125, p125, p50 and p125, p50, p75 forms will be examined for their interactions with PCNA, RFA and RFC proteins. The hope is to elucidate the functions of the various subunits. Dr. Lee has found that the enzyme interacts with PCNA and SV40 T antigen. Using a peptide competition assay the approximate location of PCNA interaction site on p125 is already known. Site directed mutagenesis will be performed. Mutant PCNA will be purified from expression clones and their ability to stimulate polymerase delta will be examined in vitro thus establishing and further dissecting the biological relevance of the PCNA - polymerase delta interaction. The RFC protein is believed to interact with PCNA and load the toroidal form onto the primer terminus. The dimer-trimer-toroid equilibrium of PCNA may influence the loading onto the primer terminus. Using cross linkers and gel permeation chromatography, these forms of PCNA will be studied in a polymerase delta - PCNA complex, in solution and on a primer-template model substrate. Using limited amount of RFC available, Dr. Lee will then study RFC - PCNA polymerase delta interaction and the possible role of RFC to influence the equilibrium of the oligomeric states of PCNA. The detection techniques that will employ labeled protein, Western blotting and phosphor imaging should allow the experiments to proceed inspite of the limited amounts of RFC being available at this time. The cell cycle dependent regulation of DNA replication is an important topic and Dr. Lee has discovered cell cycle dependent phosphorylation of polymerase delta. The proposed experiments to explore this aspect of the proposal involve mapping phosphorylation sites, studying PCNA - polymerase delta -cdk - cyclin complexes and identification of phosphatases that will dephosphoylate polymerase delta. Both known phosphatases expressed in expression clones and possible unknown ones in extracts of HeLa cells will be studied. Thus a picture of which CdK- Cyclin complex phosphorylates polymerase delta and which phosphatase(s) removes the phosphate will be known. The activity of the promoter in serum stimulated transfected cells will be analyzed. It is reassuring to note that as in other replication protein encoding genes and in the DHFR gene, the promoter of polymerase delta contains a E2F binding motif.
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