We propose to study the enzymology of base excision DNA repair in normal and regenerating rat liver. Specifically, we intend to delineate the biochemical mechanism involved in the repair of uracil residues in DNA. Uracil does not normally occur as a component of DNA, but arises from chemical deamination of cytosine or misincorporation of deoxyuridine triphosphate during DNA synthesis. Accumulation of uracil residues in the genome may result in mutagenic, cytotoxic and lethal cellular effects. We propose to purify to homogeneity, uracil-DNA glycosylase, the enzyme which initiates base excision-repair by hydrolysis of the glycosidic bond between uracil and the deoxyribose backbone of DNA. Uracil-DNA glycosylase(s) isolated from quiescent and regenerating liver will be extensively characterized to determine subcellular distribution, molecular weight, peptide structure and mode of action. The general properties of the enzyme species will be compared, which should add to understanding the relationship between the constitutive and proliferation-induced activity. An assay will be developed using DNA sequencing techniques to assess the influence of local DNA sequences and topological constraints on the formation of uracil residues produced by environmental mutagens. This aspect of the study will focus on the bisulfite catalyzed cytosine deamination reaction. Bisulfate represents the neutral aqueous form of sulfur dioxide, a major air pollutant, found in physiological fluids. The accessibility of bisulfite-produced uracil residues to uracil-DNA glycosylase will be determined for various DNA sequences. Moreover, the distribution of these lesions and accessibility to DNA repair will be demonstrated using chromatin DNA substrates. Finally, we will attempt to reconstitute a complete in vitro base excision-repair system using homologous regenerating rat liver enzymes on a uracil-containing chromatin substrate. The goal of this phase of the research is to systematically determine the mode of action of enzymes involved in base excision-repair of chromatin. It is hoped that an understanding of the biochemical mechanism of DNA repair will be basic and relevant to understanding mutagenesis and carcinogenesis.
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