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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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Pelroy, Richard
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Harvard University
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Wang, Lan; Chakravarthy, Srinivas; Verdine, Gregory L (2017) Structural Basis for the Lesion-scanning Mechanism of the MutY DNA Glycosylase. J Biol Chem 292:5007-5017
Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L (2015) Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase. J Biol Chem 290:17096-105
Pakotiprapha, Danaya; Jeruzalmi, David (2013) Small-angle X-ray scattering reveals architecture and A?B? stoichiometry of the UvrA-UvrB DNA damage sensor. Proteins 81:132-9
Sung, Rou-Jia; Zhang, Michael; Qi, Yan et al. (2013) Structural and biochemical analysis of DNA helix invasion by the bacterial 8-oxoguanine DNA glycosylase MutM. J Biol Chem 288:10012-23
Qi, Yan; Nam, Kwangho; Spong, Marie C et al. (2012) Strandwise translocation of a DNA glycosylase on undamaged DNA. Proc Natl Acad Sci U S A 109:1086-91
Crenshaw, Charisse M; Nam, Kwangho; Oo, Kimberly et al. (2012) Enforced presentation of an extrahelical guanine to the lesion recognition pocket of human 8-oxoguanine glycosylase, hOGG1. J Biol Chem 287:24916-28
Sung, Rou-Jia; Zhang, Michael; Qi, Yan et al. (2012) Sequence-dependent structural variation in DNA undergoing intrahelical inspection by the DNA glycosylase MutM. J Biol Chem 287:18044-54
Qi, Yan; Spong, Marie C; Nam, Kwangho et al. (2010) Entrapment and structure of an extrahelical guanine attempting to enter the active site of a bacterial DNA glycosylase, MutM. J Biol Chem 285:1468-78
Bowman, Brian R; Lee, Seongmin; Wang, Shuyu et al. (2010) Structure of Escherichia coli AlkA in complex with undamaged DNA. J Biol Chem 285:35783-91
Lee, Seongmin; Verdine, Gregory L (2009) Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase. Proc Natl Acad Sci U S A 106:18497-502

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