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

ABSTRACT 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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    Frontiers in Plant Science 09/2015; 6. DOI:10.3389/fpls.2015.00635 · 3.95 Impact Factor
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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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    ABSTRACT: AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.
    Nucleic Acids Research 06/2014; 42(12). DOI:10.1093/nar/gku497 · 9.11 Impact Factor
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    • "It was argued in the literature that mismatching the adenosine with cytosine would facilitate the flip-out mechanism to bring the adenosine inside the catalytic pocket (2,18). This brings up the idea of a specific recognition of the orphan counter base similar to that found for the DNA methyltransferase HhaI (19). Unfortunately, until today there is only a crystal structure of the empty deaminase domain of ADAR2 available, thus there is no structural evidence that supports this idea (18). "
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