DETERMINATION OF METHYLATION SPECIFICITY OF DSAV METHYLTRANSFERASE BYA SIMPLE BIOCHEMICAL METHOD

We have developed a simple new method that can identify the base methy lated by a sequence-specific DNA methyltransferase and have used it to identify the cytosine that is methylated by DsaV methyltransferase (M .DsaV) within its recognition sequence 5'-CCNGG. The method utilizes t he fact that exonuclease III of E.coli does not degrade DNA ends with 3' overhangs and cannot hydrolyze a phosphorothioate linkage. DNA dupl exes containing phosphorothioate linkages at specific positions were m ethylated with M.DsaV in the presence of [methyl-H-3] S-adenosylmethio nine and were subjected to exonuclease III digestion. The pattern of [ methyl-H-3] dCMP release from the duplexes was consistent with the met hylation of the internal cytosine in CCNGG, but not of the outer cytos ine. To establish the accuracy of this method, we confirmed the known specificity of EcoRII methyltransferase by the method. We also confirm ed the specificity of M.DsaV using an established biochemical method t hat involves the use of a type IIS restriction enzyme. Methylation of CCWGG (W = A or T) sequences at the internal cytosines is native to E. coli and is not restricted by the modified cytosine restriction (Met) systems. Surprisingly, the gene for M.DsaV was significantly restricte d by the McrBC system. We interpret this to mean that M.DsaV may occas ionally methylate at sequences other than CCNGG or may occasionally me thylate the outer cytosine in its recognition sequence.