The long-term goal is to determine structures of site-specific and stereospecific adducts of styrene and butadiene oxides in oligodeoxynucleotides. This information will be correlated with the results of site-specific mutagenesis and replication studies. The styrene-butadiene combination represents an important model for analyzing the consequences of human exposures to mixtures of genotoxins. The structural work will be done in the context of oligodeoxynucleotides duplexes that contain codons 11, 12, and 13, and 60, 61 and 62 of the human n-ras protoncogene. These are the ras12 and ras61 sequences, respectively. in codons 12 and 61, mutations cause activation of the protooncogene. In the next phase of this program, work with styrene oxides will be extended to mono- and cross-linked adducts of butadiene. Butadiene is observed to be of greater genotoxicity; the reasons are not well understood. Its adducts could induce less perturbation of DNA and hence be less easily recognized and repaired. Alternatively, its ability to cross-link DNA could be important. The PI will determine structures for mono adducts of butadiene and guanine N2, and adenine N1 and N6, and compare these with the corresponding styrene oxide structures. The C1 diol epoxide adducts of butadiene induce mutations in a stereospecific manner; this may occur by site-specific alterations in DNA base pairing geometry, thus enabling low levels of incorrect nucleotide insertion during DNA replication. The PI will determine whether N1 adducts cause rotation of the modified base about the glycosyl bond, thus facilitating the Dimroth rearrangement to the corresponding N6 adduct or deamination to inosine. Structure-activity relationships will be examined using NMR (SAR by NMR) in an effort to understand why adenine N6 adducts are readily bypassed by DNA polymerases whereas guanine N2 adducts are not. The C1 mono epoxide adducts of butadiene will be used as models to predict DNA cross-linking sites by butadiene diepoxide. Both intra and inter-strand crosslinks will be examined. These may cause site-specific bending or kinking of DNA. Alternatively, the 4-carbon linker of butadiene may be the optimal length to minimize DNA distortion, and potentially damage recognition. DNA sequence effects upon styrene oxide and butadiene oxide adducts will be examined. High field NMR spectroscopy will be the primary technique used to obtain structural information. Structural refinement will be accomplished using molecular dynamics/simulated annealing algorithms, restrained by nuclear Overhauser (NOE) and scalar coupling data obtained from NMR.
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