Hhal Methyltransferase Flips Its Target Base Out of the DNA Helix

W. M. Keck Structural Biology Laboratory, Cold Spring Harbor, New York 11724.
Cell (Impact Factor: 32.24). 02/1994; 76(2):357-69. DOI: 10.1016/0092-8674(94)90342-5
Source: PubMed


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.

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    • "eophilic attack on carbon 6 of cytosine in DNA . This nucleophilic attack activates an original inert carbon 5 . Abstraction of the proton at the C5 position followed by β elimination allows reformation of the C5 – C6 double bond and releases the active enzyme and DNA with a methylated cytosine ( Santi et al . , 1983 , 1984 ; Wu and Santi , 1987 ; Klimasauskas et al . , 1994 ; Peräkylä , 1998 ; Liutkeviciute et al . , 2011 ) ."
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    • "Described for the first time in calf thymus [56], methylation of cytosine is the most abundant DNA modification and occurs mainly during cellular differentiation [57]. DNA methylation is catalyzed by a well characterized family of enzymes, termed DNA methyltransferases [58]. In human cells, 60–80% of all CpG sites are highly methylated with some variation by cell type [59] [60]. "
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    • "In our model, Gln209 might make up for the lack of major groove H-bonds to the orphan guanine, when the (g)5hmC is flipped, by H-bonding with the guanine from the minor groove, instead. The two-loop mechanism used by mUHRF1 for substrate-recognition and base-flipping, in which the DNA is approached from opposite major and minor-groove directions, is also used by DNA 5mC-methyltransferases (53–55), DNA 5mC-dioxygenases (15,56), and DNA repair enzymes (57) including thymine DNA glycosylase which excises 5caC (58–60), an oxidation product of 5mC (12,13). "
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