Mechanisms of Nucleic Acid Enzymes - Pol Beta, HIV-1 Reverse Transcriptase and Mammalian Base Repair Enzymes

National Institute Of Environmental Health Sciences

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Abstract

SUMMARY OF WORK: Our research on mammalian DNA polymerase beta (pol beta) and HIV-1 reverse transcriptase (RT) has pioneered the use of a coordinated approach of structural studies (x-ray crystallography, NMR, and spectroscopy), biochemical studies, and mammalian genetic studies to understand genomic stability in mammalian cells and the mechanism of DNA polymerase action. This approach has allowed us to establish the cellular role(s) of pol beta in mammalian base excision repair. And, the approach has allowed us to establish a solid framework for future studies 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 repair with cellular checkpoint control and also with apoptosis signalling, coordination of deoxyribose phosphate removal (lyase activity) with DNA synthesis, the fidelity of DNA synthesis, the fidelity of overall DNA base excision repair, pyrophosphoroylsis and DNA lesion bypass. Rational drug design, targeting one or more of these features will allow us to strategically regulate base excision repair with pol beta specific drugs. Such agents may be useful in cancer chemotherapy and in helping us to better understand the role of DNA repair in oncogenesis and other chronic diseases. Detailed structure-function relationship studies of other base excision repair (BER) enzymes and accessory factors, such as FEN-1, PARP-1, XRCC1, DNA ligases I and III, AP endonuclease 1, 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 de-regulate base excision repair in cells. This will have implications for chemotherapy and for understanding the role of DNA repair in preventing disease after exposure to environmental toxicants. Our research also is relevant to understanding the mechanism of action of HIV-1 reverse transcriptase and in particular to knowledge of the synthesis and removal of replication blocks (i.e., chain terminators). This enzyme is capable of evolving to efficiently remove blocks to viral genome replication that are introduced during therapy with the chain terminator class of drugs, such as dideoxynucleotides. These blocks are removed by reversal of the regular DNA synthesis process termed pyrophosphorolysis. Our original studies of the mechanism of DNA synthesis and of pyrophosphorolysis in the pol beta system have allowed us to develop a framework for research on modulating the forward and reverse reactions in HIV-1 RT. Thus, our work and network of collaborators on developing pol beta specific inhibitors has established approaches toward developing potential new drugs for targeting the HIV-1 RT.