Cancer is one of today's major medical problems. The fact that some forms of cancer can be attributed to mutagenizing agents has increased the interest in determining the mechanisms by which DNA lesions are processed by cells. Much of the work on DNA repair and mutagenesis has suffered, however by the lack of pure, well characterized DNA lesions for use in enzymatic and biological studies. The object of this proposal is to synthesize the major photoproducts of TpT sites: the cis-syn, trans-syn, and (6-4) products, and sequence specifically incorporate them into oligonucleotides, restriction fragments and viruses in order to determine and correlate their physical properties with their biological ones. Our approach consists of four major phases. (1) Derivatives of the cis-syn, trans-syn and (6-4) TpT photodimers suitable for solution and solid phase DNA synthesis methodologies will be synthesized and then incorporated into single and double stranded oligonucleotides. (2) The lesion containing oligonucleotides will be analyzed by 2-D NMR techniques and molecular mechanics calculations in order to determine how they affect DNA structure. (3) These lesion containing oligonucleotides will then be incorporated into viruses and used to transform E. Coli hosts. The mutational spectrum for a given lesion will be determined by utilization of hybridization probes. (4) In order to probe the amount of distortion induced by a particular lesion, endlabelled restriction fragments containing the lesions will be prepared and studied by a combination of chemical and enzymatic probes. By embarking on this program we hope to clarify much previous work and gain new insight into mechanisms of mutagenesis and carcinogenesis. Our ultimate goal is to be able to predict the biological consequences of a lesion from its structure. If possible this could guide the screening of potential carcinogens and lead to the design of new anticancer drugs.
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