Abstract

The overall theme of this project is the enzymology of the base excision repair of oxidative damage to DNA. The enzyme 5-hydroxymethyluracil-DNA glycosylase (hmdUDG) has been purified 10,000 fold from calf thymus and primary amino acid sequence will be obtained. Primers will be constructed based on the sequence and the cDNA isolated. This will be followed by genomic cloning to make a knock out mouse. There is evidence that there are several DNA repair enzymes directed against thymine glycol residues in DNA. We have devised a technique based on the structure of the enzyme substrate complexes of these enzymes to isolate them in essentially a one step fashion. They will then be sequenced, cloned and knock out mice constructed. Similar enzymes are present in S. cerevesiae and we will isolate them, obtain their sequence and make single and double deletion mutants to evaluate their in vivo role.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA016669-23
Application #
2732956
Study Section
Special Emphasis Panel (ZRG2-MEP (02))
Program Officer
Okano, Paul
Project Start
1978-08-01
Project End
2001-06-30
Budget Start
1998-07-01
Budget End
2001-06-30
Support Year
23
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
New York University
Department
Pathology
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016

  Publications

Ocampo, Maria T A; Chaung, Wenren; Marenstein, Dina R et al. (2002) Targeted deletion of mNth1 reveals a novel DNA repair enzyme activity. Mol Cell Biol 22:6111-21
Boorstein, R J; Cummings Jr, A; Marenstein, D R et al. (2001) Definitive identification of mammalian 5-hydroxymethyluracil DNA N-glycosylase activity as SMUG1. J Biol Chem 276:41991-7
Marenstein, D R; Ocampo, M T; Chan, M K et al. (2001) Stimulation of human endonuclease III by Y box-binding protein 1 (DNA-binding protein B). Interaction between a base excision repair enzyme and a transcription factor. J Biol Chem 276:21242-9
Chheda, A D; Teebor, G W; Cunningham, R P (2000) Identification, characterization, and purification of DNA glycosylase/AP lyases by reductive crosslinking to 2'-deoxyribooligonucleotides containing specific base lesions. Methods 22:180-7
Hilbert, T P; Chaung, W; Boorstein, R J et al. (1997) Cloning and expression of the cDNA encoding the human homologue of the DNA repair enzyme, Escherichia coli endonuclease III. J Biol Chem 272:6733-40
Hilbert, T P; Boorstein, R J; Kung, H C et al. (1996) Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide. Biochemistry 35:2505-11
Zuo, S; Boorstein, R J; Cunningham, R P et al. (1995) Comparison of the effects of UV irradiation on 5-methyl-substituted and unsubstituted pyrimidines in alternating pyrimidine-purine sequences in DNA. Biochemistry 34:11582-90
Zuo, S; Boorstein, R J; Teebor, G W (1995) Oxidative damage to 5-methylcytosine in DNA. Nucleic Acids Res 23:3239-43
O'Donnell, R E; Boorstein, R J; Cunningham, R P et al. (1994) Effect of pH and temperature on the stability of UV-induced repairable pyrimidine hydrates in DNA. Biochemistry 33:9875-80
Lustig, M J; Cadet, J; Boorstein, R J et al. (1992) Synthesis of the diastereomers of thymidine glycol, determination of concentrations and rates of interconversion of their cis-trans epimers at equilibrium and demonstration of differential alkali lability within DNA. Nucleic Acids Res 20:4839-45

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