The broad, long-term objective of the proposed research is to understand the molecular basis of cellular transformation and its relationship to a mutator phenotype. We will test the hypothesis that a mutator phenotype and/or aberrant base excision repair lead to cellular transformation.
The specific aims are: (1) To test the hypothesis that Pol beta cancer-associated mutants are sufficient to transform cells, (2) To characterize the biochemical properties of Pol beta enzyme variants associated with significant human cancers, and (3) To characterize the mechanism by which Pol beta cancer -associated mutants transform cells. DNA polymerase beta (Pol B) is a eukaryotic enzyme that has a central role in cellular base excision repair (BER); Pol beta fills gaps in DNA resulting from the excision of damage. BER is a major DNA repair pathway that is responsible for the removal of an estimated 10,000 DNA lesions per cell per day. Several mutations have been identified within the Pol beta gone in prostate, lung, breast, bladder, and colon carcinomas. We will characterize the phenotypes of each of the Pol beta-cancer associated mutants biochemically and in cell lines, to determine if these mutants encode enzymes that catalyze inaccurate DNA synthesis or result in aberrant BER. Specifically, we will express the Pol beta cancer-associated mutants in various cell lines and characterize their ability to form foci, determine whether mutated forms of Pol beta induce tumors in mice, purify each of the variant proteins and study the biochemical properties of the enzyme variants using in vitro mutagenesis and kinetic assays, and characterize the mechanisms associated with cellular transformation.
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