Our long term goal is to understand, at the molecular level how human cells respond to DNA alkylation damage. It is becoming increasingly clear that organisms separated by enormous evolutionary distances employ similar proteins t protect against damage relentlessly inflicted upon their DNA, and we now know that E. coli, yeast and human cells induce the expression of specific sets of genes in responses to DNA damage. Our studies on the response of E. coli, yeast and human cells to DNA alkylation damage have become intertwined and are being executed in an integrated fashion.
Our aim i s to understand the biology, biochemistry and genetics of the responses of these cells to DNA alkylation damage. Much of this project is based upon our findings that bacterial DNA repair functions can operate in eukaryotic cells, and that the reverse also appears to be true. More specifically, E. coli Ada DNA methyltransferase can rescue human cells from the various toxic effects of DNA alkylation, a yeast DNA glycosylase and possibly a human DNA alkylation. In the main part of this application we propose to exploit and extend these observations to investigate the role of the E. coli AlkB protein, and eukaryotic AlkB analogues, in protecting cells against alkylation toxicity. We propose to isolate and characterize a S. cerevisiae gene and a human cDNA whose expression can suppress the alkylation sensitivity of E. coli alkB mutants. In addition we will express the E. coli AlkB protein in alkylation sensitive human tissue culture cells and various alkylation sensitive strains of S. cerevisiae, to determine how this protein affects the alkylation sensitive phenotype of these cells. Further, we propose to study the function of another bacterial repair methyltransferase, DNA methyltransferase II, in E. coli, S. cerevisiae and human cells. This project will contribute to an understanding of some of the events that may lead to carcinogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Program Projects (P01)
Project #
5P01ES003926-08
Application #
3840703
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Memisoglu, A; Samson, L (2000) Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe. J Bacteriol 182:2104-12
Wyatt, M D; Samson, L D (2000) Influence of DNA structure on hypoxanthine and 1,N(6)-ethenoadenine removal by murine 3-methyladenine DNA glycosylase. Carcinogenesis 21:901-8
Opperman, T; Murli, S; Smith, B T et al. (1999) A model for a umuDC-dependent prokaryotic DNA damage checkpoint. Proc Natl Acad Sci U S A 96:9218-23
Hickman, M J; Samson, L D (1999) Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Proc Natl Acad Sci U S A 96:10764-9
Li-Sucholeiki, X C; Khrapko, K; Andre, P C et al. (1999) Applications of constant denaturant capillary electrophoresis/high-fidelity polymerase chain reaction to human genetic analysis. Electrophoresis 20:1224-32
Bennett, R A (1999) The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis. Mol Cell Biol 19:1800-9
Ekstrom, P O; Borresen-Dale, A L; Qvist, H et al. (1999) Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE). Biotechniques 27:128-34
Glassner, B J; Posnick, L M; Samson, L D (1998) The influence of DNA glycosylases on spontaneous mutation. Mutat Res 400:33-44
Glassner, B J; Rasmussen, L J; Najarian, M T et al. (1998) Generation of a strong mutator phenotype in yeast by imbalanced base excision repair. Proc Natl Acad Sci U S A 95:9997-10002
Masuda, Y; Bennett, R A; Demple, B (1998) Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product. J Biol Chem 273:30352-9

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