Since changes of DNA sequence have profound genetic consequences, it is important to understand the mechanisms for maintaining and replicating these sequences if we are to understand fully the basis of genetic and degenerative diseases and possible targets for their treatment. The DNA polymerases are obviously key players in DNA metabolism and it is the applicant's goal to understand how these enzymes contribute to maintaining genetic integrity. Several years ago this group identified and purified a DNA polymerase (pol epsilon) that served to mediate DNA repair synthesis in permeabilized diploid human fibroblasts. However, it has now been suggested that in yeast, pol epsilon participates in DNA replication as well as DNA repair. However, while the """"""""core"""""""" pol epsilon from yeast and HeLa have similar catalytic, 260 kDa subunits, they have unrelated accessory subunits. To understand the role of pol epsilon in mammalian cells, this laboratory very recently produced a panel of monoclonal antibodies to HeLa pol epsilon. In so doing, subsets of monoclonal antibodies to p260, the other """"""""core"""""""" subunit, p55, and also to peptides which were shown to complex pol epsilon (p85, p70, p49) were obtained. p85 and p70 were identified as Ku autoantigen, a protein implicated in recombination and recombination repair. Using the antibodies, p55 will be sequenced, cloned and over-expressed jointly with p260. p49 will be identified and cloned. The significance of pol epsilon being complexed to Ku protein will be studied and whether the p350 protein kinase (which forms the Ku-associated DNA dependent protein kinase) also complexes to pol epsilon will be ascertained. From these results it is hoped to learn whether and how pol epsilon might be involved in a stress-response to DNA damage (e.g., might it monitor DNA during replication and call for a response, or might it be recruited for repair by such agents RNA pol II?). The monoclonal antibodies will also be used collaboratively to discern roles of human pol epsilon in nucleotide excision repair, recombinational repair, mismatch repair, replication and checkpoint control. A replication bypass assay will determine a possible role for pol epsilon in damage bypass replication. Finally a novel, extramitochondrial HeLa DNA polymerase with properties similar to both pols beta and gamma will be characterized with special attention to whether it is a homologue of yeast pol beta or a nuclear form of pol gamma.
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