DNA is constantly exposed to endogenous and exogenous agents that cleave the biopolymer and produce lesions by modifying its nucleobases. The presence of these lesions in DNA in vivo is associated with aging, diseases such as cancer, and other genetically based diseases. The overall goal of the proposed research is to probe the effects of the formamidopyrimidines (Fapy), an important family of nucleic acid lesions, on the structure and function of DNA. Fapy lesions are produced from the purines (dA, dG) by a variety of oxidative stress mechanisms. Despite their importance, the effects of the Fapy lesions on the function of DNA at the molecular level are not well understood. We recently reported the first chemical synthesis of oligonucleotides containing these lesions and their C-nucleoside analogues. In vitro experiments involving polymerase and repair enzymes by us have uncovered very interesting, potentially biologically important properties of the Fapy lesions. For instance, we have observed similarities and differences to the closely related 8-oxopurines in how the Fapy lesions interact with the Fpg repair protein. We have a so detected that both Fapy lesions decrease the fidelity of the DNA polymerase Klenow exo-, resulting in an increased level of dA misincorporation. In unpublished in vivo experiments we have shown that Fapy*dG is significantly more mutagenic in E. coli than OxodG is in the same assay. During the next funding period we request funds to continue in vitro and in vivo studies of the effects of the Fapy lesions on DNA polymerase and repair enzymes. New research directions include studying the effects of the nucleotide triphosphates of the formamidopyrimidines on polymerase and repair enzymes, and the development of fluorescent based sensors, which will detect these lesions at specific sites in DNA Increased understanding of the effects of the Fapy lesions on nucleic acid structure and function will be useful for understanding the association between nucleic acid damage and aging, as well as the etiology of diseases such as cancer.
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