Spontaneous damage of DNA bases is a major source of cancer-causing mutations. Given the thousands of lesions generated per genome every day, it is remarkable that cancer remains a relatively infrequent event with the majority of cases arising relatively late in life. With increasing life expectancy and exposure to exogenous DNA damaging agents, society bears the ever increasing cost of diagnosing and treating cancer. At the cellular level the ability to safeguard against these spontaneous lesions relies largely on the base excision repair (BER) pathway whereby DNA glycosylases scan the genome to locate and excise base lesions. The action of an apurinic (AP)-specific endonuclease, AP-lyase/DNA polymerase, and DNA ligase are required to complete repair of the DNA. Our long-term goals are to understand how BER proteins locate and selectively act on a wide range of DNA lesions within genomic DNA and how the dynamics of protein-protein and protein-DNA interactions enable coordination of multi-step, multi-enzyme repair pathways. Recent evidence suggests that nucleotide flipping, the process by which a nucleotide is extracted from the DNA duplex and bound in an active site pocket, provides much of the selectivity in distinguishing damaged and undamaged bases. We propose to test this hypothesis by directly observing flipping of damaged and undamaged nucleotides by DNA glycosylases (Aim 1). The genomic search for rare lesions is facilitated by the examination of many nucleotides with each DNA binding event, therefore we will characterize the ability of BER enzymes to move along DNA and measure the efficiency with which sites of damage are productively engaged during a scanning encounter (Aim 2). As DNA repair intermediates are potentially cytotoxic or mutagenic, it is critical that initiated BER events be completed. We propose to investigate the dynamics of protein-protein interactions in BER and determine their functional significance in the coordination of multiple enzymatic activities (Aim 3). By combining the results from pre-steady state enzyme kinetics, fluorescence spectroscopy, and structure-activity relationships we have a unique opportunity to dissect the protein-DNA dynamics important for damage recognition and repair. As BER is a critical component of the cellular defense against cancer, and because these pathways are antagonistic toward some DNA damaging agents used in the treatment of cancer, these studies have the potential to contribute both to our understanding of mutagenesis and to advances in cancer therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Macromolecular Structure and Function C Study Section (MSFC)
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Okano, Paul
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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Hedglin, Mark; Zhang, Yaru; O'Brien, Patrick J (2015) Probing the DNA structural requirements for facilitated diffusion. Biochemistry 54:557-66
Admiraal, Suzanne J; O'Brien, Patrick J (2013) DNA-N-glycosylases process novel O-glycosidic sites in DNA. Biochemistry 52:4066-74
Hedglin, Mark; Zhang, Yaru; O'Brien, Patrick J (2013) Isolating contributions from intersegmental transfer to DNA searching by alkyladenine DNA glycosylase. J Biol Chem 288:24550-9
Baldwin, Michael R; O'Brien, Patrick J (2012) Defining the functional footprint for recognition and repair of deaminated DNA. Nucleic Acids Res 40:11638-47
Hendershot, Jenna M; Wolfe, Abigail E; O'Brien, Patrick J (2011) Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase. Biochemistry 50:1864-74
Zhao, Boyang; O'Brien, Patrick J (2011) Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends. Biochemistry 50:4350-9
Hedglin, Mark; O'Brien, Patrick J (2010) Hopping enables a DNA repair glycosylase to search both strands and bypass a bound protein. ACS Chem Biol 5:427-36
Lyons, Derek M; O'Brien, Patrick J (2010) Human base excision repair creates a bias toward -1 frameshift mutations. J Biol Chem 285:25203-12
Admiraal, Suzanne J; O'Brien, Patrick J (2010) N-glycosyl bond formation catalyzed by human alkyladenine DNA glycosylase. Biochemistry 49:9024-6
Baldwin, Michael R; O'Brien, Patrick J (2010) Nonspecific DNA binding and coordination of the first two steps of base excision repair. Biochemistry 49:7879-91

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