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 multi-dimensional approach of structural (cryo-EM, fluorescence spectroscopy, NMR, and X-ray crystallography), biochemical, kinetic (transient state, pre-steady state and steady state) and mammalian genetic studies to understand human genomic stability and the mechanism and roles of DNA polymerase function. This approach has allowed us to establish the cellular role(s) of pol beta in nuclear and mitochondrial base excision repair (BER). Additionally, the approach has allowed us to establish a solid framework for rationale analysis of molecular attributes of pol beta in such important endpoints as cellular response to genotoxicants, DNA repair capacity in the context of nucleosomes (nuclear) and nucleoids (mitochondria), coordination of DNA repair with cellular checkpoint control and apoptosis signaling, coordination of enzymatic activities (deoxyribose phosphate lyase and DNA synthesis), the fidelities of DNA synthesis and overall BER, and DNA lesion bypass. Rational drug design, targeting one or more of these features permit us to strategically regulate BER with pol beta specific drugs. Such agents are useful in chemotherapies and have provided insights into the role of DNA repair in oncogenesis and other chronic diseases. Development of specific inhibitors or other modulators for BER enzymes will allow strategic de-regulation of BER in cells. This will have implications for chemotherapy and for understanding the role of DNA repair in preventing disease after exposure to environmental toxicants. Since we have recently identified pol beta in mitochondrial DNA maintenance, pol beta cellular trafficking will impact cellular response to oxidative stress and highlights the importance of mitonuclear communication. Our research also is relevant to understanding the mechanism of action of HIV-1 reverse transcriptase (RT) 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 azidothymidine. These blocks are removed by reversal of the regular DNA synthesis process termed pyrophosphorolysis and result in viral drug resistance. Our original studies of the mechanism of the forward DNA synthesis reaction and the reverse pyrophosphorolysis reaction with the pol beta have allowed us to develop a framework for research in modulating the forward and reverse reactions in HIV-1 RT. Thus, our work with developing pol beta specific inhibitors has established approaches toward developing potential new drugs for targeting the HIV-1 RT. In the past year, we have expanded our studies of structure-function relationship of DNA polymerases to include kinetic and X-ray crystallographic studies of other pol beta-related polymerases lambda and mu. These studies will define unique attributes that can be exploited in inhibitor design to increase target specificity.