) The broad, long-term objective of the cell proposed research is to understand the molecular basis of cellular transformation and its relationship to a mutator phenotype. The approach we will take in this project is to test the hypothesis that aberrant base excision repair leads to cellular transformation.
The specific aims are: (1) To characterize the biological and biochemical properties of DNA polymerase b enzyme variants associated with significant human cancers; (2) To test the hypothesis that mutant DNA polymerase b enzymes have the ability to interfere with base excision repair or to confer a mutator phenotype on mammalian cells; (3) To test the hypothesis that expression of the Pol b mutant enzymes is associated with cellular transformation. DNA polymerase b (Pol b) is a eukaryotic enzyme that has a central role in cellular base excision repair (BER); Pol b 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 b gene in prostate and colon carcinomas. We will characterize the phenotypes of each of the Pol b-cancer associated mutants biochemically and in cell lines, to determine if these mutants encode enzymes that catalyze in accurate DNA synthesis or result in aberrant BER. Specifically, we will purify the mutant proteins and study their DNA binding properties and fidelity using in vitro assays. We will also determine whether these variants confer a mutator phenotype on mammalian cells and if expression of these variants results in aberrant BER. Finally, we will ascertain whether expression of the cancer-associated Pol b mutants results in cellular transformation and tumorigenesis by examining focus formation, growth in soft agar, and tumor formation and metastasis in nude mice in response to expression of the Pol b variants. The results obtained from these studies have the potential to further our understanding of the molecular basis of cellular transformation and, therefore, neoplastic disease.
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