In vitro specificity of EcoRI DNA methyltransferase.

The sequence selectivity of enzyme-DNA interactions was analyzed by comparing discrimination between synthetic oligonucleotides containing the canonical site GAATTC and altered DNA sequences with the EcoRI DNA methyltransferase. The specificities (kcat/KmDNA) are decreased from 5- to 23,000-fold relative to the unmodified site. For several substrates the decrease in kcat makes a disproportionate contribution to the specificity difference, suggesting that discrimination is mediated by the placement of critical catalytic residues rather than binding interactions. This is supported by our observation that specificity changes are generally not followed by changes in the stability of the methyltransferase-DNA complexes. Also, base pair substitutions near the site of methylation result in greater decreases in complex stability, suggesting that recognition and catalytic mechanisms overlap.