The mutagenicity of 5-hydroxymethyluracil (HmUra) in the DNA of Chinese hamster V79 cells will be characterized. A HGPRT- V79 cell line containing the bacterial gpt gene (V79gpt) will be developed. The formation of gpt- cells following exposure to HmdUrd will be quantified, and representative gpt- clones will be isolated for genetic analysis. Southern analysis will be used to determine whether HmdUrd induced gpt- cells have large deletions in the gpt gene. Direct sequencing using the polymerase chain reaction will determine whether gpt- cells have point mutations. Our ability to introduce a single DNA base modification, the HmUra residue, will be further exploited to study the role of poly(ADP-ribose) in base excision repair. The effects of HmUra in DNA on poly(ADP-ribose) synthesis, NAD depletion, and poly(ADP-ribose) polymerase activity will be measured. It will be determined whether the toxic effects of 3- aminobenzamide (3AB) to cells containing HmUra in their DNA result from the inhibition of poly (ADP-ribose) synthesis. It will also be determined whether 3AB increases the mutagenicity of HmdUrd and whether 3AB alters the spectrum of mutations produced by HmdUrd. Finally, to test the hypothesis that the toxicity of HmUra residues in DNA results primarily from their repairability, V79 cells will be chemically mutagenized and HmUrd will be used to select HmdUrdr mutants. HmdUrdr cells which have lost HmUra-DNA glycosylase activity will be characterized 1) to determine whether the cells have an increased, decreased or unchanged spontaneous mutation rate at the gpt locus, as compared with the parent cell, 2) to determine whether these cells are more susceptible to mutagenesis by HmUrd or ionizing radiation and 3) to determine whether such cells are resistant to the synergistic toxic effects of HmdUrd and 3AB.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA051060-04
Application #
3459692
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1990-01-01
Project End
1994-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
New York University
Department
Type
Schools of Medicine
DUNS #
004514360
City
New York
State
NY
Country
United States
Zip Code
10012
Mi, L J; Chaung, W; Horowitz, R et al. (2001) Excessive base excision repair of 5-hydroxymethyluracil from DNA induces apoptosis in Chinese hamster V79 cells containing mutant p53. Carcinogenesis 22:179-86
Mi, L J; Mahl, E; Chaung, W et al. (1997) Lack of phenotypic alteration of hmUra-DNA glycosylase-deficient hamster cells exposed to DNA-damaging agents. Mutat Res 374:287-95
Chaung, W; Boorstein, R J (1997) Molecular spectrum of mutations induced by 5-hydroxymethyl-2'-deoxyuridine in (CHO)-PL61 cells. Mutat Res 373:125-37
Chaung, W; Mi, L J; Boorstein, R J (1997) The p53 status of Chinese hamster V79 cells frequently used for studies on DNA damage and DNA repair. Nucleic Acids Res 25:992-4
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
Boorstein, R J; Chiu, L N; Teebor, G W (1992) A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase is resistant to the toxic effects of the thymidine analog 5-hydroxymethyl-2'-deoxyuridine. Mol Cell Biol 12:5536-40
Boorstein, R J; Hilbert, T P; Cunningham, R P et al. (1990) Formation and stability of repairable pyrimidine photohydrates in DNA. Biochemistry 29:10455-60