Project 1 is concerned with the chemical biology of the aristolochic acids (AAs), a series of naturally-occurring nitrophenanthrene carboxylic acids, and their related lactams. The toxic effects of AAs lead to chronic renal disease and urothelial cancer. Chemical synthesis will provide large quantities of the AAs, their lactams, and related compounds for further chemical modification, metabolic investigations and toxicity studies. The known dA(N6)- and dG(N2)-adducts found in mammalian DNA will be synthesized and incorportated site-specifically into oligodeoxynucleotides with sequences containing known mutagenic """"""""hot spots"""""""" in the ras and p53 genes. The goals of the project are to characterize the biological properties of AA-adducts in duplex DNA and to correlate the mechanisms of lesion repair and mutagenesis with the three dimensional structure. Sophisticated 2D NMR experiments coupled with restrained molecular dynamics calculations will provide solution structures for AA-adducts in duplex DNA. Additionally, the stabilities of these lesion-containing duplexes will be determined by UV-melting profiles. Nucleotide excision repair (NER) is the only known mechanism for repair of bulky lesions, and mechanisms for damage in closed circular plasmids containing a single dA(N6)- or dG(N2)-adduct. While AA is known to induce AT to TA transversions, the biochemical mechanisms involved have not been determined. We will examine the efficiency and fidelity of translesion synthesis (TLS) to identify the DNA polymerase(s) responsible for error-free and error-prone synthesis. Correction of the mechanisms of damage recognition, repair of AA-adducts and their miscoding and mutational specificity with three dimensional structure and duplex stability will provide a molecular basis for the nephrotoxic and carcinogenic properties of aristolochic acids.
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