OF WORK: Understanding the relationships between structure and function for a DNA enzyme holds the promise of allowing us to develop specific inhibitors and other modulators of the enzyme's activity through rational design approaches. Although this general idea is now fundamental in the field of structural biology, we are still in the earliest stages of bringing the approach to its full potential toward drug development for cancer therapy, AIDS treatment and treatment of other viral diseases. Our studies of mammalian DNA polymerase beta have pioneered the use of a coordinated approach of structural studies (x-ray crystallography, NMR, and spectroscopy), biochemical studies and mammalian genetic studies. This approach has allowed us to establish the cellular roles of DNA polymerase beta in mammalian base excision repair. And, the approach has allowed us to establish a solid framework for future studies of the role of individual amino acid residues in this enzyme in such important endpoints as cellular response to genotoxicants, the rate of DNA repair, coordination of DNA synthesis with DNA ligation, coordination of deoxyribose phosphate removal (lyase activity) with DNA synthesis, the fidelity of DNA synthesis, the fidelity of overall DNA base excision repair, and DNA lesion bypass. Rational drug design, targeting these endpoints will allow us to strategically regulate base excision repair with DNA polymerase beta specific drugs. Such medicines will be useful in cancer chemotherapy and in helping us to better understand the role of DNA repair in oncogenesis.Detailed structure-function relationship studies of other base excision repair enzymes, such as XRCC1, DNA ligases I and III, AP endonuclease, and the various DNA glycosylases, will be undertaken in the future. Development of specific inhibitors or other modulators for these enzymes will allow us to strategically deregulate base excision repair in cells. This could have implications for chemotherapy and for understanding the role of DNA repair in preventing disease especially after exposure to environmental toxins.
Aims and Accomplishments: We have established the cellular role of mammalian DNA polymerase in single-nucleotide base excision repair. We have discovered the structure of DNA polymerase complexed with its biological substrates, single-nucleotide gapped DNA and incoming dNTP, both matched and mismatched, and we have improved the understanding of the mechanism of """"""""templating"""""""" by this enzyme. We have reconstituted single-nucleotide base excision repair in vitro using purified human enzymes. We have discovered an alternate base excision repair pathway in mammalian cells, termed long patch base excision repair, and we have reconstituted portions of this pathway using purified human enzymes. We have characterized protein-protein partnerships necessary for the long patch pathway.
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