Differential stabilization of reaction intermediates: specificity checkpoints for M.EcoRI revealed by transient fluorescence and fluorescence lifetime studies.
M.EcoRI, a bacterial sequence-specific S-adenosyl-L-methionine-dependent DNA methyltransferase, relies on a complex conformational mechanism to achieve its remarkable specificity, including DNA bending, base flipping and intercalation into the DNA. Using transient fluorescence and fluorescence lifetime studies with cognate and noncognate DNA, we have characterized several reaction intermediates involving the WT enzyme. Similar studies with a bending-impaired, enhanced-specificity M.EcoRI mutant show minimal differences with the cognate DNA, but significant differences with noncognate DNA. These results provide a plausible explanation of the way in which destabilization of reaction intermediates can lead to changes in substrate specificity.
Youngblood
Ben
B
Program in Biomolecular Science and Engineering, University of California, Santa Barbara, CA 93106-9510, USA.
Bonnist
Eleanor
E
Dryden
David T F
DT
Jones
Anita C
AC
Reich
Norbert O
NO
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009154:Mutation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)