DNA bending by EcoRI DNA methyltransferase accelerates base flipping but compromises specificity.
EcoRI DNA methyltransferase was previously shown to bend its cognate DNA sequence by 52 degrees and stabilize the target adenine in an extrahelical orientation. We describe the characterization of an EcoRI DNA methyltransferase mutant in which histidine 235 was selectively replaced with asparagine. Steady-state kinetic and thermodynamic parameters for the H235N mutant revealed only minor functional consequences: DNA binding affinity (KDDNA) was reduced 10-fold, and kcat was decreased 30%. However, in direct contrast to the wild type enzyme, DNA bending within the mutant enzyme-DNA complexes was not observed by scanning force microscopy. The bending-deficient mutant showed enhanced discrimination against the methylation at nontarget sequence DNA. This enhancement of enzyme discrimination was accompanied by a change in the rate-limiting catalytic step. No presteady-state burst of product formation was observed, indicating that the chemistry step (or prior event) had become rate-limiting for methylation. Direct observation of the base flipping transition showed that the lack of burst kinetics was entirely due to slower base flipping. The combined data show that DNA bending contributes to the correct assembly of the enzyme-DNA complex to accelerate base flipping and that slowing the rate of this precatalytic isomerization can enhance specificity.
Allan
B W
BW
Department of Chemistry and Biochemistry and Program in Biochemistry and Molecular Biology, University of California, Santa Barbara, California 93106-6081, USA.
Garcia
R
R
Maegley
K
K
Mort
J
J
Wong
D
D
Lindstrom
W
W
Beechem
J M
JM
Reich
N O
NO
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics
D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases