OF WORK: The highly conserved, ubiquitous DNA repair pathway known as base excision repair (BER) is one of the key cellular defense mechanisms against deleterious effects of metabolism and environmental exposures. For example, mutations in E. coli and mammalian BER genes alike cause cells to suffer spontaneous genomic instability and such instability upon environmental exposure. Generally, BER repaired lesions are smaller lesions in DNA bases, instead of large, bulky substitutions at bases, strand breaks or inter-strand cross-links. Among the most important sources of genomic damage repaired by BER are: hydrolytic loss of DNA bases; oxidation of bases and sugars; alkylation of DNA bases and DNA phosphate groups; and misincorporation errors during DNA replication. Recent studies in a number of laboratories, including ours, have confirmed that there are at least three sub-pathways of BER in mammalian cells. Our hypothesis is that lack of appropriate BER in mammalian cells can play a critical role in carcinogenesis and other degenerative conditions. We have recapitulated the main mammalian BER pathway in vitro using the four purified human proteins, uracil-DNA glycosylase, AP endonuclease, DNA polymerase beta and DNA ligase I. We have cloned the human and mouse genes for these four enzymes, expressed the recombinant human proteins in E. coli and insect cells, and prepared cell lines with genetic alterations in each gene. Transgenic mouse models are being prepared for study of the cellular and tissue requirements for each enzyme. This project includes studies of the cellular role of BER in DNA repair, apoptosis, mutagenesis, chromosome stability, DNA lesion bypass, cell singalling, growth control, and human and mouse carcinogenesis. In addition, recent studies by Cooper et al. and Lindhal et al. have suggested that transcription-coupled BER is an important pathway in oxidative damage DNA repair. Investigation of this pathway is underway, in collaboration with Drs. Cooper and Sancar.
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