Abstract

DNA extracted from Escherichia coli grown in the presence of 5-azacytidine (azaC-DNA) inhibits the DNA(cytosine-5)methyltransferase extracted from E. coli K12 cells without affecting the DNA(adenine-N⁶)methyltransferase present in these same cells. The inhibition is time-dependent and the rate of inhibition can be decreased by addition of substrate DNA. The inhibitory capacity of the DNA is destroyed by incubation with pancreatic deoxyribonuclease or micrococcal nuclease; however, the inhibited enzyme cannot be reactivated by treatment with these enzymes. The cytosine methylases methylate only double-stranded DNA. Similarly, the inhibitory DNA loses activity if it is heat-denatured, and regains activity upon reannealing. The DNA will also inhibit the EcoRII and HpaII modification methylases which also synthesize 5-methylcytosine in DNA. Digestion of the inhibitory DNA with the respective restriction endonuclease destroys, in part, the inhibitory activity of the DNA for the respective methylase. Base analysis indicated that 5-azacytidine replaced 8.4% of the cytosine in the azaC-DNA. These results suggest that DNA containing 5-azacytidine irreversibly inhibits DNA(cytosine-5)methylases.