We propose to continue our studies concerning the enzymology of uracil-excision DNA repair in rat liver cells. The proposal derives from previous observation that two distinct subcellular forms of uracil-DNA glycosylase have been identified. The major objectives are to elucidate the structural and functional relationships between the nuclear and mitochondrial enzymes and to attempt to isolate an enzyme complex which may function coordinately in DNA repair. We will determine whether mitochondria are capable of performing uracil-excision DNA repair in vivo. To achieve these goals we propose to complete the purification of the rat liver mitochondrial uracil-DNA glycosylase and characterize the properties of the homogeneous enzyme. Using highly purified preparations of both the nuclear and mitochondrial uracil-DNA glycosylase we will prepare two collections of monoclonal antibodies. The immunological relationship between the two forms will be determined by cross-reactivity using ELISA and Western blot analysis. Similarities in protein structure will also be determined using peptide mapping techniques. An in situ assay for detecting uracil-DNA glycosylase in SDS-polyacrylamide gels will be developed. Use of this technique will allow us to rapidly determine the distribution of various molecular weight forms of the enzyme in different subcellular fractions. We will also attempt to isolate a multi-enzyme complex that acts to perform base-excision repair. This aspect of the study will focus on, but not be limited to, the isolation of a uracil-DNA glycosylase complex using molecular sieving and immunoaffinity chromatography. A mitochondrial AP-endonuclease will be purified and the ability of endonuclease catalyzed incisions to support DNA synthesis by DNA polymerase-Gamma will be analyzed. Finally, a cell free system for measuring DNA repair in mitochondria will be developed. The goal of their phase will be to determine if mitochondria repair uracil-residues in its genome and whether different mitochondrial genes are equally susceptible to repair. It is hoped that an understanding of the mechanism of DNA repair will be basic and relevant to understanding biochemical pathways for preventing mutagenesis and carcinogenesis.
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