Abstract
Changes in DNA bending and base flipping in a previously characterized specificity-enhanced M.EcoRI DNA adenine methyltransferase mutant suggest a close relationship between precatalytic conformational transitions and specificity (Allan, B. W., Garcia, R., Maegley, K., Mort, J., Wong, D., Lindstrom, W., Beechem, J. M., and Reich, N. O. (1999) J. Biol. Chem. 274, 19269-19275). The direct measurement of the kinetic rate constants for DNA bending, intercalation, and base flipping with cognate and noncognate substrates (GAATTT, GGATTC) of wild type M.EcoRI using fluorescence resonance energy transfer and 2-aminopurine fluorescence studies reveals that DNA bending precedes both intercalation and base flipping, and base flipping precedes intercalation. 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 an approximately 2500-fold increase in the reverse rate constants for intercalation and base flipping, respectively. Thus, a 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 enzyme specificity and, in particular, sequence-specific DNA modification. Because many DNA methyltransferases and DNA repair enzymes induce similar DNA distortions, these results are likely to be broadly relevant.
Highlights
These enzymes frequently undergo significant conformational changes upon binding their cognate sequence, a clear example of an induced fit mechanism
The molecular basis of how changes in precatalytic conformational transitions can result in such a profound increase in specificity is the focus of this study
Using a FRET-based assay [18], we examined equilibrium and pre-equilibrium kinetics of binding, bending, and intercalation for the cognate sequence with the target adenine in the complement strand methylated (GAATTC) and 3 noncognate sequences as follows: A6 (GAATCC), A4 (GAATTT), and A3 (GGATTC)
Summary
These enzymes frequently undergo significant conformational changes upon binding their cognate sequence, a clear example of an induced fit mechanism. The resultant intermediates provide a theoretical basis for modulating specificity through the concept of kinetic proofreading, as proposed for several DNA polymerases [9]. These checkpoints can directly impact specificity by modifying the partitioning of reaction intermediates prior to catalysis differentially between cognate and noncognate substrates (9 –11). The schematic and kinetic mechanism of M.EcoRI presented in Scheme 1 represent the chemical and conformational transitions observed during the methylation of cognate DNA. Our results are presented in the context of kinetic proofreading concepts, which provide a quantitative framework for understanding sequence-specific DNA modification
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