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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM032823-07
Application #
3281976
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-12-01
Project End
1991-11-30
Budget Start
1989-09-01
Budget End
1989-11-30
Support Year
7
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Oregon State University
Department
Type
Earth Sciences/Resources
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339
Doublali, M; Chouaib, A; Khallouk, A et al. (2010) Primary malignant melanoma of prostate. Urol Ann 2:76-7
Chen, Cheng-Yao; Mosbaugh, Dale W; Bennett, Samuel E (2005) Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase. DNA Repair (Amst) 4:793-805
Bennett, Samuel E; Chen, Cheng-Yao; Mosbaugh, Dale W (2004) Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease. Proc Natl Acad Sci U S A 101:6391-6
Chen, Cheng-Yao; Mosbaugh, Dale W; Bennett, Samuel E (2004) Mutational analysis of arginine 276 in the leucine-loop of human uracil-DNA glycosylase. J Biol Chem 279:48177-88
Sung, Jung-Suk; Mosbaugh, Dale W (2003) Escherichia coli uracil- and ethenocytosine-initiated base excision DNA repair: rate-limiting step and patch size distribution. Biochemistry 42:4613-25
Wong, Isaac; Lundquist, Amy J; Bernards, Andrew S et al. (2002) Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a ""pinch-pull-push"" mechanism. J Biol Chem 277:19424-32
Bennett, S E; Sung, J S; Mosbaugh, D W (2001) Fidelity of uracil-initiated base excision DNA repair in DNA polymerase beta-proficient and -deficient mouse embryonic fibroblast cell extracts. J Biol Chem 276:42588-600
Sung, J S; Bennett, S E; Mosbaugh, D W (2001) Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts. J Biol Chem 276:2276-85
Sanderson, R J; Bennett, S E; Sung, J S et al. (2001) Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts. Prog Nucleic Acid Res Mol Biol 68:165-88
Sung, J S; Mosbaugh, D W (2000) Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV. Biochemistry 39:10224-35

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