Interaction of M.SssI and M.HhaI with single-base mismatched oligodeoxynucleotide duplexes

As part of an attempt to elucidate the mode of interaction of the CpG methyltransferase M.SssI with its substrate, we have prepared a series of double-stranded oligodeoxyribonucleotides containing one mismatch in the CpG recognition site. The mismatched duplexes were used to analyze the binding capabilities and enzymatic activity of M.SssI and M.HhaI (recognizes GCGC). We demonstrate here that M.SssI binds specifically to substrates containing either a C/A or G/T mismatch in the recognition sequence, i.e., 5'-GCGC/CACG-5' or 5'-GCGC/CGTG-5', respectively. The enzyme also shows significant enzymatic activity with these mismatched substrates. These results suggest that site recognition and methylation by M.SssI take place on the same strand. M.HhaI bound and methylated the C/A mismatch very efficiently, but recognition of the G/T mismatch was scarcely detectable.