DNA bending by EcoRI DNA methyltransferase accelerates base flipping but compromises specificity

EcoRI DNA methyltransferase was previously shown to bend its cognate DNA se quence by 52 degrees and stabilize the target adenine in an extrahelical or ientation. We describe the characterization of an EcoRI DNA methyltransfera se mutant in which histidine 235 was selectively replaced with asparagine. Steady-state kinetic and thermodynamic parameters for the H235N mutant reve aled only minor functional consequences: DNA binding affinity (K-D(DNA)) wa s reduced 10-fold, and k(cat) was decreased 30%. However, in direct contras t to the wild type enzyme, DNA bending within the mutant enzyme-DNA complex es was not observed by scanning force microscopy, The bending-deficient mut ant showed enhanced discrimination against the methylation at nontarget seq uence DNA. This enhancement of enzyme discrimination was accompanied by a c hange in the rate-limiting catalytic step. No presteady-state burst of prod uct formation was observed, indicating that the chemistry step (or prior ev ent) had become rate-limiting for methylation. Direct observation of the ba se flipping transition showed that the lack of burst kinetics was entirely due to slower base flipping. The combined data show that DNA bending contri butes to the correct assembly of the enzyme-DNA complex to accelerate base flipping and that slowing the rate of this precatalytic isomerization can e nhance specificity.