This Program Project seeks to understand mechanisms by which exposures to environmentally- and endogenously-produced bis-electrophiles, such as vinyl chloride, acrolein, crotonaldehyde, and 4- hydroxynonenal (4-HNE), induce genotoxic response, ultimately impacting human health. All organisms must successfully maintain a stable genome in the face of continuous insults arising from genotoxic chemicals. Adduction of DNA by these aldehydic bis-electrophiles yields interstrand crosslinks, interstrand crosslinks, DNA-protein conjugates, and various regioisomeric mono-adducts. These compounds likely contribute to background levels of inter- and /intrastrand crosslinks and DNA-protein crosslinks formed in human cells. The inability to repair genomic damage correlates with human disease-e.g., cancer, premature aging, fatty liver disease, and atherosclerosis. The three projects utilize complementary technologies to address common goals-understanding the mutagenic and cytotoxic consequences of DNA adduction by bis-electrophiles. Emphasis will be placed upon elucidation of the chemistry and biology of (1) interstrand and (2) intrastrand crosslinks formed by bis-electrophiles, and (3), examining the chemistry and biology of potentially highly mutagenic regioisomeric adducts formed by bis-electrophiles-particularly at N1-dA and N3-dC, and N7-dG, as compared to adducts formed at N2-dG. Project 1, led by Dr. Carmelo Rizzo, will develop novel synthetic routes to construct oligodeoxynucleotides containing site- and stereospecific inter- and intrastrand DNA crosslinks, and N1-dA and N3-dC, and N7-dG adducts of bis-electrophiles, will investigate the in vitro replication bypass and secondary chemical reactivity of these adducts, and will work to identify these adducts in cellular DNA. Project 2, led by Dr. Stephen Lloyd, will generate biological endpoints with respect to replication, repair, and mutagenesis in response to the introduction of these lesions into cells. The essential steps required for repair of interstrand crosslinks in the absence of homologous recombination will be determined. The mutagenic spectra of intrastrand crosslinks, and of N1-dA and N3-dC adducts will be determined in mammalian cells. The role of DNA sequence in modulating the mutagenic spectra will be examined in mammalian cells, with emphasis on frameshift-prone sequences. The biological processing of interstrand crosslinks will be examined. Project 3, led by Dr. Michael Stone, will provide structural data for oligodeoxynucleotides containing inter- and intrastrand crosslinks using a combination of NMR and X-ray crystallographic approaches. The interactions of modified DNA templates with bypass polymerases will be determined. Interactions between the three projects will be iterative, with biological data from Project 2 focusing synthetic priorities in Project 1 and structural priorities in Project 3. Project 1 will provide novel adducts for the biological and structural studies, and access to DNA modified with these adducts, via the DNA Synthesis Core Facility. ?
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