Analysis of conformational transitions that facilitate DNA methyltransferase specificity and catalysis

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 and/or base flipping by an enzyme, can provide further discrimination between cognate and noncognate substrates by the enzyme. Using fluorescence resonance energy transfer (FRET) and 2-Aminopurine (2AP) fluorescence studies with the cognate sequence (GAATTC) for WT M.EcoRI, the temporal relationship between DNA bending, intercalation of the DNA substrate by the enzyme, and base-flipping is elucidated. Destabilization of these intermediates provides a molecular basis for understanding how conformational transitions contribute to specificity. The 3500 and 23,000-fold decreases in sequence specificity for noncognate sites GAATTT and GGATTC are accounted for largely by a ∼2500 fold increase in the reverse rate constants for intercalation and base flipping, respectively. The predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis.; Building upon the concepts that were developed with the M.EcoRI studies, an improvement in sequence specificity of the DNA cytosine methyltransferase HhaI is achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments with 2AP show that mutations disrupting this interface interfere with catalysis by destabilizing the extrahelical "flipped" cytosine base in the active site. This destabilization, caused by a double mutant, results in a ∼500-fold specificity enhancement compared to the WT enzyme.; To better understand the contribution of specific active site atoms to the catalytic enhancement of M.HhaI and the rearrangement of these atoms to facilitate the chemistry step, three crystal structures were solved using the true cofactor S-adenosyl-L-methionine (pre-chemistry), and the cofactor product S-adenosyl-L-homocysteine (post-chemistry).; Much work remains in resolving how discrimination occurs for eukaryotic DNA methyltransferases in comparison to prokaryotic DNA methyltransferases mentioned above. To facilitate our advancement in understanding eukaryotic DNA methyltransferases, a model system to explore substrate discrimination in cells was developed. Using HIV-1, the mechanism for over-expression and targeting of the mammalian DNA methyltransferase 1 (DNMT1) to specific sites in the genome has been probed.