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.
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