The long term goal of the proposed work is to understand the molecular biological and biophysical processes that lead to the production of mutations when mutagen-damaged DNA is replicated. This proposal aims to exploit the analytical power of experiments with vectors that carry a single defined and specifically located UV photoproduct. These tools will be used (i) to investigate the question of how DNA photoproduct structure is related to its mutagenic properties, (ii) to analyze the different components of the spectrum of UV-induced mutations, and attempt to reconstruct this spectrum from its component parts, (iii) to determine whether double-stranded DNA provides opportunities for error-free bypass of lesions, in addition to error-prone translesion replication, and (iv) to explore and define the roles of various normal and mutant genes in bypass and mutagenesis. These studies are likely to advance our understanding of UV mutagenesis, and of mutagenic mechanisms in general. UV is a significant environmental carcinogen, one that is likely to become of even greater importance in the future because of ozone depletion in the upper atmosphere, and mutagenesis is an important step in cancerogenesis. A better understanding of mutagenic mechanisms will help in estimating health risks from carcinogens, particularly in the difficult issues of extrapolation from high acute to low chronic doses, and from one species to another. The experimental design for this proposal is to construct vectors that carry a single uniquely placed mutagenic photoproduct, and determine the properties of the lesion when the vector is replicated in vivo. Estimates will be made of the three basic parameters that define a lesion's mutagenic potential: frequency of bypass, error-frequency of bypass, and mutation spectrum. A variety of photoproducts will be examined, and also a variety of vectors, including both single- and double-stranded constructs. Photoproducts to be studied include cis-syn and trans-syn cyclobutane dimers, and both the normal and dewar isomers of pyrimidine (6-4) pyrimidone adducts, each located in a variety of bipyrimidine target sites, and flanked by a variety of different nucleotides.
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