Binding of the EcoRll methylase to azacytosine-containing DNA

Binding of DNA(oytoaine-5)methyltransferases to azaoytosine containing DNA is stimulated by the presence of S-adenosyl-methionine or its analogs slnefungin or S-adenosyl-L-homo-cysteine. Methylation of the DNA is therefore not necessary for binding to occur. There Is no relationship between the affinity of the analog for the EooRII enzyme and its ability to Btinulate binding. The DNA-enzyme oomplex partially dissociates on incubation in 0.1J sodium dodeoyl sulfate and 0.5 H ammonium aoetate. Some of this DNA oould again form a tight complex with enzyme, indica-ting that DNA-enzyme oomplex formation is reversible. Binding ooours when the seoond oytosine in the sequence CCAGG is substituted by azaoytoslne. This is the oytosine that would normally be methylated by the enzyme. The binding is there-fore due to specific interaction of the raethylase with azacyto-sine at the site it would normally methylate. INTRODOCTION. 5-azaoytosine-oontalnlng DNA (azaC-DNA) Inhibits bacterial DNA-(oytoslne-5)methyl transf erases (1). The SLM. coll liflFII modi-fication metbylase, whioh is a cytosine metbylase, is inhibited irreversibly. Irreversibi 1 lty is defined in tMs instance by the inability to regenerate enzyme activity by dilution of the en-zyme-DNA oomplex, or by digesting the DNA with either microooccal nuolease or DNase I after the enzyme complex is formed. Both these enzymes destroy the inhibitory ability of the DNA if they are inoubated with the DNA before addition of the methylase (1). Azacytosine-containing DNA fragments end labelled with ^? bind to the purified EcoRII methylase. For binding to occur the DNA must also contain the sequence recognized by the £c_o_RII methylase, the sequence CCA/TGG (2). It is the second C from the 5 ' end that is methylated by this enzyme (3). Binding can be demonstrated by polyacry1 amide gel eleotrophoresis of the comple