Direct real time observation of base flipping by the EcoRI DNA methyltransferase.
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 k(methylation) 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.
Allan
B W
BW
Department of Chemistry, University of California, Santa Barbara 93106-6081, USA.
Beechem
J M
JM
Lindstrom
W M
WM
Reich
N O
NO
D004247:DNA / Q000378:metabolism*; D004795:Enzyme Stability; D007536:Isomerism; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*
D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)