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Conformational transitions as molecular determinants of specificity for the DNA methyltransferase EcoRI

DNA modifying enzymes have evolved a delicate balance between sequence specificity and efficient catalysis. Utilization of indirect readout of a DNA sequence, such as DNA bending by an enzyme, provides further discrimination beyond the initial binding event between the enzyme and DNA. Changes in the DNA bending and base flipping transitions in a previously characterized specificity‐enhanced mutant M. EcoRI DNA adenine methyltransferase suggest a close relationship between precatalytic conformational transitions and specificity. We present fluorescence resonance energy transfer (FRET) and 2‐AminoPurine (2AP) fluorescence studies with the cognate sequence (GAATTC) for WT M. EcoRI, and reveal a temporal relationship between DNA bending, intercalation of the DNA substrate by the enzyme, and base‐flipping. The direct measurement of kinetic rate constants for DNA bending, intercalation, and base‐flipping with cognate and non‐cognate substrates (GAATTT, GGATTC) by WT M. EcoRI, provide a molecular understanding of how these conformational transitions impact on specificity. The 3500 and 23,000‐fold decreases in sequence specificity of WT M. EcoRI for noncognates GAATTT and GGATTC are accounted for largely by a ~2500 fold increase in the reverse rate constants for intercalation and base flipping. The predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis. Our results provide a basis for understanding sequence‐specific DNA modification. Research funding provided by NSF.