Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases

The SinI and EcoRII DNA methyltransferases recognize sequences (GG(A)/TCC a nd CCA/(T)GG, respectively), which are characterized by an A/T ambiguity. R ecognition of the A-T and T-A base pair was studied by in vitro methyltrans ferase assays using oligonucleotide substrates containing a hypoxanthine-C base pair in the central position of the recognition sequence. Both enzymes methylated the substituted oligonucleotide with an efficiency that was com parable to methylation of the canonical substrate. These observations indic ate that M.SinI and M.EcoRII discriminate between their canonical recogniti on site and the site containing a G-C or a C-G base pair in the center of t he recognition sequence (GG(G)/CCC and CCG/(C)GG, respectively) by interact ion(s) in the DNA minor groove. M.SinI mutants displaying a decreased capac ity to discriminate between the GGA/TCC and GG(G)/CCC sequences were isolat ed by random mutagenesis and selection for the relaxed specificity phenotyp e. These mutations led to amino acid substitutions outside the variable reg ion, previously thought to be the sole determinant of sequence specificity. These observations indicate that A/T versus G/C discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.