5-azacytidine, a nucleoside analog, is used in the treatment of acute myelogenous leukemia and experimentally in the treatment of sickle cell disease and Beta-thalassemia. The latter use resulted from the finding that the drug caused the expression of genetic material not normally expressed in tissue culture cells. This is believe to be due to its activity as a DNA methylase inhibitor. This inhibitory activity is dependent on the incorporation of azacytidine into DNA. Our objectives are to use 5-azacytidine to study and characterize the interactions of DNA-methylases with DNA as a means of determining the rules that govern specific protein-DNA interactions and to identify the lesions by which this drug leads to cell death. Azacytidine-containing DNA forms stable complexes with DNA-cytosine methylases, acting as a site specific inactivator of these enzymes. We will prepare DNA fragments containing azacytosine at specific sites in DNA and study their interaction with the bacterial EcoRII methylase. Protein binding to such fragments can be detected in crude cellular extracts, thus allowing us to readily characterize the binding specificity of mutant methylases. The binding site of the enzyme will be determined and studied by site specific mutagensis. The drug is lethal to bacteria that contain the EcoRII methylase. Since the methylase is superfluous for cell survival, and does not metabolize the drug, we believe this lethal effect to be due to stable binding of the enzyme to DNA in the cell. We will determine the basis for this effect by mutagenizing the cloned methylase and determining the relationship between methylase activity, binding to the recognition site in DNA, and the lethal effect of the drug. Mutations in DNA repair systems also decrease survival of cells treated with 5-azacytidine. The repair system(s) required for survival will be characterized. We will determine if the methylase enzyme forms a stable complex in vivo with azacytosine containing DNA and if this lesion can be repaired. The effect of the methylase bound to DNA on enzyme reactions involved in RNA synthesis, DNA synthesis, DNA initiation and recombination will be studied in vitro.
| Som, S; Friedman, S (1990) Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine. J Biol Chem 265:4278-83 |
| Lal, D; Som, S; Friedman, S (1988) Survival and mutagenic effects of 5-azacytidine in Escherichia coli. Mutat Res 193:229-36 |
| Som, S; Bhagwat, A S; Friedman, S (1987) Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme. Nucleic Acids Res 15:313-32 |
| Friedman, S (1986) Binding of the EcoRII methylase to azacytosine-containing DNA. Nucleic Acids Res 14:4543-56 |
| Friedman, S (1985) The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases. J Biol Chem 260:5698-705 |
