Research in this laboratory is concerned with the biochemistry of radiation-induced DNA damage, its repair and the mechanisms by which it is translated into biologic effects such as cell lethality, mutation, genetic recombination and carcinogenesis. Particular attention is given to the relationship of DNA damage and its repair to the clinical manifestations of certain human genetic disorders that exhibit cellular hypersensitivity to ionizing and nonionizing radiations and a predisposition to cancer. The overall goal of this proposal is to provide a better understanding of the repair and biologic significance of bifilar DNA damage, which is defined operationally as localized damage to both strands of a DNA double-helix. For the purpose of this proposal, UV-induced closely opposed cyclobutane pyrimidine dimers have been chosen as a model """"""""lesion"""""""". It is expected that the bifilar nature of closely opposed dimers will require more complicated mechanisms for repair and therefore may have greater biologic significance than dimers induced at relatively isolated positions.
The specific aims of this study are 1) to characterize more precisely factors that affect the detection of closely opposed dimers as bifilar enzyme-sensitive sites, 2) to examine the DNA sequence and dose dependence of closely opposed dimer induction, 3) to extend studies on the repair of closely opposed dimers in normal and UV-sensitive human and rodent cells, and 4) to determine the role of closely opposed dimers in the induction of genetic damage such as mutations and genetic recombination. The experimental design to be used in the pursuit of these aims will employ 1) synthetic oligodeoxynucleotides to examine the closely opposed dimer sequence dependence and to explore experimental parameters affecting their detection as bifilar ESS, 2) velocity sedimentation-based bifilar enzyme-sensitive site assays for studies on the repair of closely opposed dimers in cellular DNA of mammalian cells and 3) shuttle vector based systems to examine the role of closely opposed dimers in UV-induced mutagenesis and recombination.
