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Vol. 273, Issue 4, 2368-2373, January 23, 1998
From the Department of Chemistry and Program in Biochemistry and
Molecular Biology University of California, Santa Barbara,
California 93106-6081 and the DNA methyltransferases are excellent prototypes
for investigating DNA distortion and enzyme specificity because
catalysis requires the extrahelical stabilization of the target base
within the enzyme active site. The energetics and kinetics of base
flipping by the EcoRI DNA methyltransferase were
investigated by two methods. First, equilibrium dissociation constants
(KDDNA) were determined for
the binding of the methyltransferase to DNA containing abasic sites or
base analogs incorporated at the target base. Consistent with a base
flipping mechanism, tighter binding to oligonucleotides containing
destabilized target base pairs was observed. Second, total intensity
stopped flow fluorescence measurements of DNA containing 2-aminopurine
allowed presteady-state real time observation of the base flipping
transition. Following the rapid formation of an enzyme-DNA collision
complex, a biphasic increase in total intensity was observed. The fast
phase dominated the total intensity increase with a rate nearly
identical to kmethylation determined by rapid
chemical quench-flow techniques (Reich, N. O., and Mashoon, N. (1993) J. Biol. Chem. 268, 9191-9193). The restacking
of the extrahelical base also revealed biphasic kinetics with the
recovered amplitudes from these off-rate experiments matching very
closely to those observed during the base unstacking process. These
results provide the first direct and continuous observation of base
flipping and show that at least two distinct conformational transitions
occurred at the flipped base subsequent to complex formation.
Furthermore, our results suggest that the commitment to catalysis
during the methylation of the target site is not determined at the
level of the chemistry step but rather is mediated by prior
intramolecular isomerization within the enzyme-DNA complex.
Direct Real Time Observation of Base Flipping by the
EcoRI DNA Methyltransferase
, Department of Molecular
Physiology and Biophysics, Vanderbilt University Medical Center,
Nashville, Tennessee 37232-0615
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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