HhaI methyltransferase flips its target base out of the DNA helix.
The crystal structure has been determined at 2.8 A resolution for a chemically-trapped covalent reaction intermediate between the HhaI DNA cytosine-5-methyltransferase, S-adenosyl-L-homocysteine, and a duplex 13-mer DNA oligonucleotide containing methylated 5-fluorocytosine at its target. The DNA is located in a cleft between the two domains of the protein and has the characteristic conformation of B-form DNA, except for a disrupted G-C base pair that contains the target cytosine. The cytosine residue has swung completely out of the DNA helix and is positioned in the active site, which itself has undergone a large conformational change. The DNA is contacted from both the major and the minor grooves, but almost all base-specific interactions between the enzyme and the recognition bases occur in the major groove, through two glycine-rich loops from the small domain. The structure suggests how the active nucleophile reaches its target, directly supports the proposed mechanism for cytosine-5 DNA methylation, and illustrates a novel mode of sequence-specific DNA recognition.
Klimasauskas
S
S
W. M. Keck Structural Biology Laboratory, Cold Spring Harbor, New York 11724.
Kumar
S
S
Roberts
R J
RJ
Cheng
X
X
D044503:5-Methylcytosine; D001426:Bacterial Proteins / Q000648:ultrastructure; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000648:ultrastructure; D015257:DNA-Cytosine Methylases / Q000378:metabolism / Q000648:ultrastructure*; D006190:Haemophilus / Q000201:enzymology; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases