We measured the kinetics of
DNA bending by
M.EcoRI using
DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate
DNA site, energy transfer is decreased upon
enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different
dye pairs, when the
dye pairs are placed on the respective 3'-ends, the effect is cofactor- and
protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled
DNA shows the anticipated increased energy transfer with
EcoRV endonuclease, which also
bends this sequence, and no change in energy transfer with
EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from
M.EcoRI binding, mediated by a mechanism novel for
DNA methyltransferases, combining
DNA bending and an overall expansion of the
DNA duplex. The
M.EcoRI protein sequence is poorly accommodated into well defined classes of
DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly,
M.EcoRI has an intercalation motif observed in the FPG
DNA glycosylase family of repair
enzymes.
Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following
AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.