Spontaneous damage to the four bases of DNA is a major cause of the mutations that give rise to cancer. Most of these genetic lesions are corrected by a pathway known as base-excision DNA repair (BER). The key components of BER are DNA glycosylases, professional lesion-hunting enzymes that scan the genome in search of particular kinds of base damage, then catalyze excision of the damaged base from the DNA backbone. The long-term goals of our studies are to understand how these enzymes locate damaged bases amidst the vast excess of normal DNA, and the precise reaction pathways that they utilize. A comprehensive, fundamental understanding of DNA damage recognition and removal represents the solution to a major aspect of the tumorigenesis puzzle. In the proposed studies, we will focus on the cellular resistance to the genotoxic effects of oxidative stress. Specifically, we will study base-excision repair of the highly mutagenic lesion 8-oxoguanine (oxoG) by two DNA glycosylases, human Ogg1 protein and bacterial MutM. Here we outline a broad-based, interdisciplinary approach that employs chemical synthesis of substrate analogs, semisynthetic site-specific modification of the proteins, high-resolution structural analysis, single-molecule fluorescence spectroscopy, and in vitro biochemistry to elucidate significant unresolved issues in the structure and function of these proteins. In particular, we aim to develop a structural picture of the multistep lesion-processing reaction cascade catalyzed by Ogg1 and MutM, by stalling the reaction at various points along the way to capture static structures, and by using time-resolved X-ray to observe the base-excision reaction in real time. We propose furthermore to determine whether the base-excision process as studied in vitro is a reasonably faithful representation of repair in vivo, by using Xenopus oocyte extracts as a model for the latter. Finally, we propose to extend our understanding of structure/function in BER to include the MutY protein, which functions in eukaryotes and prokaryotes to correct the mutagenic damage resulting from misreplication of 8-oxoguanine.
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