Human cells contain singular DNA repair pathways to excise critical lesions from DNA as a mechanism to prevent the initiation of neoplasia. Alterations in these pathways may result in the failure to remove miscoding lesions thus facilitating tumor development. Alternatively, as many cancer therapeutic agents are themselves DNA damaging agents, tumor cells may utilize the identical pathways to mitigate the efficacy of cancer therapy. Accordingly, this research proposal is designed to probe the basic mechanisms through which human cells recognize and remove critical DNA adducts. Investigation of the base excision repair enzyme uracil DNA glycosylase will be used as a model system. These studies will use the highly purified normal human enzyme; natural mutant(s) of the enzyme isolated from individuals with Bloom's syndrome, a human disease characterized by a high cancer rate; and a panel of anti-human uracil DNA glycosylase monoclonal antibodies. The objectives of this proposal are: 1) To examine the immunology and sequence homology of human uracil DNA glycosylases. Enzymes structure will be examined by peptide mapping and identification of the glycosylase monoclonal antibody binding sites. The specific amino acid alteration(s) in the Bloom's syndrome enzyme will be determined and related directly to alterations in the nucleotide sequence of the human uracil DNA glycosylase gene. 2) To examine the basic mechanisms through which this miscoding lesion is removed from DNA. These investigations will include kinetic analysis of the catalytic reaction as well as the mechanism through which 5- flyorouracil, a cancer chemotherapeutic agent, inhibits glycosylase activity. These studies will use both the normal and mutant human enzymes. These experiments will define the relationship between alterations in protein structure and the ability of human DNA repair enzymes to prevent the initiation of neoplasia by the removal of critical DNA lesions.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA029414-10
Application #
3168699
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1981-02-01
Project End
1994-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
10
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Temple University
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Wang, X; Sirover, M A; Anderson, L E (1999) Pea chloroplast glyceraldehyde-3-phosphate dehydrogenase has uracil glycosylase activity. Arch Biochem Biophys 367:348-53
Sirover, M A (1996) Minireview. Emerging new functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells. Life Sci 58:2271-7
Wurzer, J C; Tallarida, R J; Sirover, M A (1994) New mechanism of action of the cancer chemotherapeutic agent 5-fluorouracil in human cells. J Pharmacol Exp Ther 269:39-43
Weng, Y; Sirover, M A (1993) Developmental regulation of the base excision repair enzyme uracil DNA glycosylase in the rat. Mutat Res 293:133-41
Mansur, N R; Meyer-Siegler, K; Wurzer, J C et al. (1993) Cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in normal human cells. Nucleic Acids Res 21:993-8
Mauro, D J; De Riel, J K; Tallarida, R J et al. (1993) Mechanisms of excision of 5-fluorouracil by uracil DNA glycosylase in normal human cells. Mol Pharmacol 43:854-7
Meyer-Siegler, K; Rahman-Mansur, N; Wurzer, J C et al. (1992) Proliferative dependent regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in human cells. Carcinogenesis 13:2127-32
Meyer-Siegler, K; Mauro, D J; Seal, G et al. (1991) A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci U S A 88:8460-4
Seal, G; Tallarida, R J; Sirover, M A (1991) Purification and properties of the uracil DNA glycosylase from Bloom's syndrome. Biochim Biophys Acta 1097:299-308
Cool, B L; Sirover, M A (1990) Proliferation-dependent regulation of DNA glycosylases in progeroid cells. Mutat Res 237:211-20

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