The recognition pathway for the DNA cytosine methyltransferase M.HhaI.
Enzymatic sequence-specific DNA modification involves multiple poorly understood intermediates. DNA methyltransferases like M.HhaI initially bind nonspecific DNA and then selectively bind and modify a unique sequence. High-resolution NMR was used to map conformational changes occurring in M.HhaI upon binding nonspecific DNA, a one base pair altered noncognate DNA sequence, and both hemimethylated and unmethylated cognate DNA sequences. Comparisons with previous NMR studies of the apoenzyme and enzyme-cofactor complex provide snapshots of the pathway to sequence-specific complex formation. Dramatic chemical shift perturbations reaching many distal sites within the protein are detected with cognate DNA, while much smaller changes are observed upon nonspecific and noncognate DNA binding. A cooperative rather than stepwise transition from a nonspecific to a cognate complex is revealed. Furthermore, switching from unmethylated to hemimethylated cognate DNA involves detectable protein conformational changes 20-30 A away from the methyl group, indicating high protein sensitivity and plasticity to DNA modification.
Zhou
Hongjun
H
Department of Chemistry and Biochemistry, University of California at Santa Barbara, Santa Barbara, California 93106-9510, USA.
Purdy
Matthew M
MM
Dahlquist
Frederick W
FW
Reich
Norbert O
NO
D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D019175:DNA Methylation / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006190:Haemophilus / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015257:DNA-Cytosine Methylases