There are many environmental chemicals classified as mutagens and/or carcinogens which damage DNA by forming exocyclic rings on nucleic acid bases. These include vinyl chloride; mucochloric acid from chlorination of drinking water; the widely administered therapeutic halonitrosoureas, e.g. BCNU and the benzene metabolites, p-benzoquinone and z,z, muconaldehyde. The mechanisms by which such damage may lead to malignant transformation is not known at the molecular level, although the majority of the population is exposed to such agents.
Our specific aims are directed toward understanding how such endogenous chemicals alter DNA structure and lead to biological endpoints, such as mutation and, ultimately, cancer. All these chemicals modify bases in DNA, forming structures which have an added 5 membered exocyclic ring between the 1 and N6 of A, the 3 and N4 of C and the 1 and N2 or the N2 and 3 of G and, in some cases, an extra functional group.
The specific aims are directed toward understanding structure-function relationships, namely how the new ring structure influences biochemical events such as replication and repair.
This aim first requires the synthesis of modified nucleosides and their phosphoramidites, which will be site-specifically incorporated into defined oligonucleotides. Oligonucleotides will be initially studied in terms of whether the modified bases allow complete replication by polymerases in vitro and, if so, what base is inserted opposite the modified base. This gives insights into possible mutation or lethality. The role of flanking bases in directing a mispair will also be investigated since, in vivo, mutations occur in specific favored sequences. Repair is also sequence- dependent. Since persistent adducts can lead to mutation if not removed prior to replication, repair of new adducts in oligomers will be tested using human cell-free extracts which contain a wide variety of repair enzymes. Concurrently, structural changes in a doublestranded oligomer, with a single modified base, can be assessed using biophysical techniques. The methods will involve chemical synthesis of precursors and preparation of defined oligomers with a single adduct, using phosphoramide chemistry. Purity of precursors will be assessed using HPLC, nmr and MS. Replication will be by a primer extension assay with 5'-32P endlabeling. Structural perturbations will be probed using thermal denaturation, calorimetry, electrophoretic mobility, enzyme probes and molecular modeling.
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