Project 3: Conformational Studies of Adducted Oligodeoxynucleotides

Vanderbilt University, Nashville TN

Investigators

Linked publications & trials

Abstract

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