STRUCTURE, FUNCTION AND EVOLUTION OF DNA REPAIR ENZYMES

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DESCRIPTION (provided by the applicant): Hydroxyl radicals form endogenously and following exposure to ionizing radiation, and produce a broad spectrum of potentially lethal and mutagenic DNA lesions that are removed by Base Excision Repair (BER) enzymes. BER has been studied extensively using naked DNA substrates, but there is virtually no direct information on how BER enzymes process free radical-induced DNA damage in nucleosomes, which are the fundamental subunit of chromatin. The twin goals of the proposed studies are to identify rate-limiting steps in the recognition and repair of oxidative lesions in nucleosomes, and to identify factors that regulate or circumvent these rate-limiting steps. To that end, we will assemble model nucleosomes that contain discretely positioned, defined lesions or DNA structures that represent intermediates in the BER pathway. These will be incubated with purified, human bifunctional DNA glycosylases and selected other BER enzymes to test HYPOTHESIS 1, that the rate-limiting step in BER of free radical-induced single base DNA lesions in nucleosomes is the lesion recognition step, which is governed by the rotational and translational position of DNA damage relative to the nucleosome dyad. We then will test HYPOTHESIS 2, that alterations in nucleosome structure permit BER glycosylases to act on lesions that would otherwise be sterically inaccessible. We will investigate the extent to which partial, reversible unwrapping of DNA from the histone octamer facilitates access to sterically occluded lesions. Lesion-containing nucleosomes will also be incubated with yeast nuclear extracts, prepared from wild type cells and mutants defective in selected BER accessory factors or chromatin remodeling agents to help guide future efforts to isolate factors that regulate BER in cells. INTEGRATION WITH OTHER COMPONENTS OF THE PROGRAM PROJECT: The currently funded projects in this Research Program focus on the structure, enzymatic properties, and substrate preferences of three families of DNA repair enzymes, two of which figure prominently in studies outlined in this proposal. Moreover these studies will substantially broaden our understanding of how human DNA glycosylases interact with lesions in nucleosomes, and will potentially reveal factors that enhance BER of lesions in chromatin. In addition to these common thematic goals, these studies will materially benefit from the bioinformatics and enzyme purification services that will be available through Cores A and B within the program project. RELEVANCE TO PUBLIC HEALTH: The information generated by the proposed studies will advance our understanding of the basic mechanisms that lead from radiation exposure to development of cancer. Additionally, radiation therapy is widely used in cancer therapy. The proposed studies will advance our understanding of factors that affect radiation sensitivity of cells. Modulation of such factors could help maximize therapeutic gain during radiotherapy.