Conformational transitions as determinants of specificity for the DNA methyltransferase EcoRI.
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.
Youngblood
Ben
B
Program in Biomolecular Science and Engineering, Department of Chemistry and Biochemistry, University of California, Santa Barbara 93106-9510, USA.
Reich
Norbert O
NO
D016880:Anisotropy; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000502:physiology*; D013696:Temperature; D013997:Time Factors
D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)