This is a FIRST application which seeks to gain a detailed understanding of the proteins and mechanisms involved in human base excision repair. Preliminary data suggests that DNA polymerase b (Polb) is the major base excision repair polymerase in HeLa extracts, but the known properties of Polb suggest that other factors too must be involved. The goal is to eventually reconstitute the complete repair process with purified components. Specifically, the investigator proposes to (1) use an in vitro system employing double-stranded uracil-containing oligonucleotides to isolate the core enzymes (glycosylases, polymerases, nucleases, ligases) required for base excision repair from fractionated HeLa extracts; (2) to isolate additional proteins/factors involved in base excision repair, for example, additional proteins necessary to repair chromatin as opposed to naked DNA, or accessory proteins which stimulate or regulate base excision repair; (3) to characterize the purified repair proteins and mechanisms involved in base excision repair and identify the reaction intermediates they produce; and (4) to investigate the role that base excision repair defects may play in human carcinogenesis. A defect in base excision repair would be expected to lead to increased mutation rates and to a higher cancer incidence. The possibility that defective base excision repair may be involved in hereditary or sporadic cancers will be investigated. Cell lines (Bloom's, syndrome, XRCC1 mutants, 46BR, premature aging syndrome cells, Xeroderma pigmentosum) derived from cancer-prone and/or mutagen-sensitive individuals will be used to make extracts. These extracts will be tested in a quantitative manner for their ability to support base excision repair. If significant defects are found, the follow-up will include determining which repair protein is altered. The relevance to human health is based on the hypothesis that base excision repair plays a significant role in the prevention of cancer. By providing a more detailed understanding of the base excision repair pathway and directly testing for defects in cancer cells, the importance of this type of repair in preventing carcinogenesis should be clarified.
