The adduction of DNA by the environmentally and endogenously produced genotoxins vinyl chloride, acrolein, crotonaldehyde, and 4-hydroxynonenal (4-HNE), yields /nte/strand crosslinks, /nfrastrand crosslinks, DNA-protein conjugates, and regioisomeric mono-adducts. These chemicals likely contribute to background levels of inter- and /nfrastrand crosslinks and DNA-protein crosslinks in human cells. An inability to repair genomic damage correlates with human disease?e.g., cancer, premature aging, fatty liver disease, and atherosclerosis. Using materials prepared by Project 1 and the DNA Synthesis Core, this project will utilize NMR and crystallography to obtain high resolution structural data for DNA modified with these bis-electrophiles, to delineate the underlying structural basis for their mutagenicity and cytotoxicity. It will be determined how formation of /nterstrand crosslinks depends upon the identity and stereochemistry of substituents at C6 of proximal 1,N2-dG enal adducts. Using reduced crosslinks of the 6R- and 6S crotonaldehyde adducts it will be determined if the 6S crosslink creates a greater structural perturbation to DNA than does the 6R crosslink. The (6S,8R,11S) crosslink of the 4-HNE adduct possessing the same absolute stereochemistry as the 6R-crotonaldehyde crosslink will be characterized as to its chemistry. Work with reduced peptide-DNA crosslinks will focus on the conformation of the glycosyl torsion angle, which will orient the peptide in the minor vs. major groove. Our work will be correlated with that in Project 2 designed to understand crosslink repair. The chemistry of intrastrand crosslinks will be determined via incorporation of NMR-active isotopes. Using saturated analogs of these crosslinks, it will be determined if the intervening T in the 5'-GTX-3'sequence extrudes from the duplex. Residual dipolar coupling measurements in partially oriented samples, and gel electrophoretic mobility assays, will probe crosslink-induced DNA bending. The capacity of the Sulfolobus solfataricus Dpo4 polymerase to accommodate these crosslinks will be examined with crystallography of binary (Pol + DNA) and ternary (Pol + DNA + dNTP) complexes. The results will be correlated with site-specific mutagenesis experiments in Project 2. NMR studies will examine the hypothesis that all but one of the stereoisomeric 4-HNE adducts exist largely as 1 ,N2-dG adducts in the syn conformation about the glycosyl bond. Likewise, N1-dA and N3-dC adducts, and their N1-dl and N3-dU deamination products, may exist in the syn conformation about the glycosyl bond. Crystallography involving binary and ternary complexes of human Pol-iota and Pol-kappa with modified primer-template complexes will probe the sequential bypass of enal adducts by these polymerases, as observed by Project 2. It will be determined if hPol-iota exploits the syn conformations of these adducts for bypass. The role of hPol-kappa in positioning the primer 3'-OH to catalyze extension following insertion by hpol-iota will be determined. The potential for strand slippage during bypass of these lesions by Pol-eta, leading to frameshifts, will be determined.
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