DNA polymerase delta is a central enzyme in DNA replication and repair. The goals of the first aim are to identify, clone, and express the as yet unidentified subunits of mammalian pol delta that together with the core enzyme (125 kda and 50 kDa) constitute the holoenzyme. Pol delta holoenzyme complexes have been isolated and peptide sequences of candidate polypeptides will be obtained to permit cloning of the cDNAs. Parallel approaches to isolating these cDNAs will be pursued by yeast two hybrid screening and affinity chromatography. The recombinant subunits will be used to reconstitute the holoenzyme which will then be characterized.
The second aim i s to map the protein binding sites between PCNA and pol delta by site-directed mutagenesis. The design of the mutants will be based on the involvement of the interdomain connector loop of PCNA in binding pol delta. The binding of the N2 region of pol delta to these sites will be investigated by the use of model peptides and site directed mutagenesis. These studies will define the interactions between pol delta and PCNA, and the extent to which p21/Waf1 binding sites overlap those of pol delta.
The third aim i s to establish the role of cyclin/cdks in control of pol delta by the use of specific inhibitors of the cdks. The phosphorylation of purified pol delta by the cyclin-cdks in control of pol delta y the use of specific inhibitors of the cdks. The phosphorylation of purified pol delta by the cyclin-cdks will be analyzed in vitro to determine the specificity of the different cyclin/cdks, the stoichiometry of phosphorylation, the sites of phosphorylation, and the functional effects of phosphorylation. The interactions of pol delta, PCNA and the cyclin/cdks will be studied to identify and characterize the complexes that are formed in order to establish the physical basis for a hypothesis that the cyclin binding to PCNA may be a targeting mechanism.
The fourth aim i s to study the transcriptional regulation of POLD1 gene by the tumor suppressor p53. The effects of p53 on the expression of the gene for the p125 subunit of pol delta (POLD1) will be studied to confirm the novel finding that p53 represses the activity of the POLD1 promoter. The studies will identify and confirm the identify of a putative p53 consensus binding sequence in the pol delta promoter. The goals of this work are highly health- related, as the replication of the genome and the maintenance of its integrity are central to life processes. Knowledge of these processes are critical to an understanding of disease processes, particular in cancer. These studies may also lead to future therapeutic drug design based on the molecular understanding of the protein-protein interactions that control DNA replication.
Showing the most recent 10 out of 65 publications
