Although butadiene is a known mutagen and suspected carcinogen, the exact identity of the DNA adducts that are responsible for these events are not known. Furthermore, no studies have been conducted that ascertain the DNA repair mechanisms that are responsible for the removal of butadiene-modified nucleotides. In order to address these fundamental issues of butadiene-induced mutagenecity, as part of a prior collaboration with the Department of Chemistry at Vanderbilt University, oligonucleotides have been synthesized that contain site and stereospecific butadiene adducts: the R and S stereochemistries of the mono and diepoxides at N2-guanine and N6-adenine, as well as N6-N6 adenine and N2-N2-guanine intrastrand cross-links. The availability of these reagents facilitates the study of the molecular mechanisms of butadiene-induced mutagenesis and repair. This will be accomplished by engineering these modified DNAs into larger DNA templates that are suitable for detailed in vitro polymerization studies; these will include analyses of prokaryotic and eukaryotic replicative and repair polymerases. These same DNAs will be engineered into shuttle vectors that are designed for in vivo replication in either prokaryotic or eukaryotic systems. Mutagenic spectrum and frequencies will be assessed using differential hybridization strategies that are amenable to very high through-put of vectors. Additionally, collaborations between the Lloyd and Van Houten laboratories will establish which of the butadiene-modified DNAs are subject to correction by nucleotide excision repair using the E. coli UvrABC complex as the prototype. Fur collaborations are planned between the Lloyd/Van Houten groups with the Albrecht and Muganda groups, that are designed to measure rates of repair in cells exposed to butadiene metabolites using quantitative PCR assays. Finally, butadiene- induced DNA damage and repair will be measured in DNA repair proficient and deficient mice that have been exposed to butadiene gas; this study is in collaboration with Dr. Ward.
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