This multidisciplinary research program, focussing on the genetic toxicology of DNA adducts, is comprised of six projects and a core facility. Oligodeoxynucleotides, modified at preselected sites by adducts and related lesions are prepared, using solid-state automated DNA synthesis techniques or by post-synthetic modification. Participants in the Program utilize these oligomers to explore the biological consequences of lesions introduced at predefined positions in DNA. Projects include: 1. Synthesis of Oligodeoxynucleotides Bearing Mutagens as Site-Specified Adducts; 2. Mechanisms of Chemical Mutagenesis; 3. Effect of DNA Adducts on Transcription by Eukaryotic RNA Polymerases; 4. Studies on DNA Polymerase Alpha and Delta; 5. Immunocytochemical Studies on DNA Adducts, and 6. Molecular Modeling of DNA Adducts and Other Lesions. During the past two years, a variety of site-specifically modified oligodeoxynucleotides have been prepared. Lesions include aminofluorene derivatives, propanodeoxyguanosine (acrolein) model adducts and synthetic abasic sites. New shuttle plasmid vectors have been developed which replicate in bacteria and mammalian cells. In this experimental system, single lesions induce mutagenic responses targeted to the site of chemical modification. Synthetic abasic sites were used to explore the detailed mechanism of action of DNA polymerase alpha. These lesions block incorporation of deoxynucleotide triphosphates by DNA polymerase alpha by inhibiting binding of the template-primer. An in vitro DNA repair system of eukaryotes has been characterized--the first cell-free system of this type to be described. New immunocytochemical techniques have been developed. Chemists associated with this Program will develop new synthetic methods to facilitate introduction of adducts, site-specifically, into DNA. Site-specific mutagenesis will be applied to several DNA adducts and related lesions suspected of playing a role in the genotoxicity of environmental mutagens. Major enzymatic activities that comprise the repair process will be purified and characterized in extracts prepared from human cells and X. laevis. DNA polymerase alpha and delta will be studied mechanistically, using templates modified with specific adducts. Monoclonal and polyclonal antibodies will be raised against chemically-defined oligodeoxynucleotide-protein conjugates containing abasic sites or DNA adducts. Immunocytochemical methods will be used to localize and quantitate adducts in tissues and cellular DNA. Molecular model building, energy minimization and two-dimensional NMR spectroscopic techniques will be combined in an attempt to correlate molecular structure of DNA adducts with the effects of these modifications on relevant biological processes. Taken together, these studies should provide important insights into the molecular toxicology of DNA adducts.
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