A causative role has been established between compromised DNA repair and the development of human cancers. DNA bases are subject to oxidative stress undergoing chemical modification of guanine and other bases as a result of endogenous oxidants, inflammatory responses to infection and injury, and exposure to redox-active environmental toxins. While much attention has been focused on G to T mutations as a result of the oxidized guanine lesion 8-oxoguanine (OG), this project investigates the hyperoxidized guanine lesions resulting from further oxidation of OG. These lesions constitute a family of highly mutagenic hydantoin structures including spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) that appear to cause G to C in addition to G to T mutations. In the present work, related members of this class of compounds will be studied to further explore the relationship between oxidation pathway, product structure, and activity with DNA processing enzymes including base excision repair and nucleotide excision repair mechanisms. The new structures to be studied include amine adducts of oxidized guanosine. Novel pathways are proposed that might explain additional observations in the mutagenic spectrum of oxidative stress, including the formation of double-stranded tandem lesions.
The specific aims of this work are to: (1) resolve structural questions surrounding the formation of hydantoin lesions in duplex DNA through the use of synthetic lesion-containing oligodeoxynucleotides, mass spectrometry, and x-ray crystallography, (2) understand the roles of base excision repair vs. nucleotide excision repair for hydantoin lesions via in vitro and in vivo biochemical assays with a particular focus on the role of the Nei-like (hNEIL1) and Fpg glycosylases, and (3) develop innovative assays to detect hydantoin products in cell lysates by generating aptamers to DNA lesions coupled to fluorescent readouts.
Oxidative stress plays a significant role in the damaging DNA creating lesions that underlie cancer, aging, neurological and cardiac disorders. This project will help define how specific oxidized DNA bases and base adducts lead to mutations related to cancer. In addition, methods will be developed to monitor the formation and excision of these damaged bases from DNA, revealing new information about the relationship between DNA damage and cancer.
|Eckenroth, Brian E; Fleming, Aaron M; Sweasy, Joann B et al. (2014) Crystal structure of DNA polymerase ? with DNA containing the base lesion spiroiminodihydantoin in a templating position. Biochemistry 53:2075-7|
|Rowland, Meng M; Schonhoft, Joseph D; McKibbin, Paige L et al. (2014) Microscopic mechanism of DNA damage searching by hOGG1. Nucleic Acids Res 42:9295-303|
|Krokeide, Silje Z; Laerdahl, Jon K; Salah, Medya et al. (2013) Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroimindiohydantoin and guanidinohydantoin. DNA Repair (Amst) 12:1159-64|
|Fleming, Aaron M; Orendt, Anita M; He, Yanan et al. (2013) Reconciliation of chemical, enzymatic, spectroscopic and computational data to assign the absolute configuration of the DNA base lesion spiroiminodihydantoin. J Am Chem Soc 135:18191-204|
|Fleming, Aaron M; Burrows, Cynthia J (2013) G-quadruplex folds of the human telomere sequence alter the site reactivity and reaction pathway of guanine oxidation compared to duplex DNA. Chem Res Toxicol 26:593-607|
|McKibbin, Paige L; Fleming, Aaron M; Towheed, Mohammad Atif et al. (2013) Repair of hydantoin lesions and their amine adducts in DNA by base and nucleotide excision repair. J Am Chem Soc 135:13851-61|
|Zhou, Jia; Liu, Minmin; Fleming, Aaron M et al. (2013) Neil3 and NEIL1 DNA glycosylases remove oxidative damages from quadruplex DNA and exhibit preferences for lesions in the telomeric sequence context. J Biol Chem 288:27263-72|
|McKibbin, Paige L; Kobori, Akio; Taniguchi, Yosuke et al. (2012) Surprising repair activities of nonpolar analogs of 8-oxoG expose features of recognition and catalysis by base excision repair glycosylases. J Am Chem Soc 134:1653-61|
|Sejersted, Yngve; Hildrestrand, Gunn A; Kunke, David et al. (2011) Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxia-ischemia. Proc Natl Acad Sci U S A 108:18802-7|
|Fleming, Aaron M; Muller, James G; Ji, Insun et al. (2011) Characterization of 2'-deoxyguanosine oxidation products observed in the Fenton-like system Cu(II)/H2O2/reductant in nucleoside and oligodeoxynucleotide contexts. Org Biomol Chem 9:3338-48|
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