Hhal Methyltransferase Flips Its Target Base Out of the DNA Helix
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 ) ."
ABSTRACT: Somatic embryogenesis (SE) is a powerful tool for plant genetic improvement when used in combination with traditional agricultural techniques, and it is also an important technique to understand the different processes that occur during the development of plant embryogenesis. SE onset depends on a complex network of interactions among plant growth regulators, mainly auxins and cytokinins, during the proembryogenic early stages, and ethylene and gibberellic and abscisic acids later in the development of the somatic embryos. These growth regulators control spatial and temporal regulation of multiple genes in order to initiate change in the genetic program of somatic cells, as well as moderating the transition between embryo developmental stages. In recent years, epigenetic mechanisms have emerged as critical factors during SE. Some early reports indicate that auxins and in vitro conditions modify the levels of DNA methylation in embryogenic cells. The changes in DNA methylation patterns are associated with the regulation of several genes involved in SE, such as WUS, BBM1, LEC, and several others. In this review, we highlight the more recent discoveries in the understanding of the role of epigenetic regulation of SE. In addition, we include a survey of different approaches to the study of SE, and new opportunities to focus SE studies.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). "
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). "
ABSTRACT: Adenosine deaminases that act on RNA (ADAR) are a class of enzymes that catalyze the conversion of adenosine to inosine in RNA. Since inosine is read as guanosine ADAR activity formally introduces A-to-G point mutations. Re-addressing ADAR activity toward new targets in an RNA-dependent manner is a highly rational, programmable approach for the manipulation of RNA and protein function. However, the strategy encounters limitations with respect to sequence and codon contexts. Selectivity is difficult to achieve in adenosine-rich sequences and some codons, like 5′-GAG, seem virtually inert. To overcome such restrictions, we systematically studied the possibilities of activating difficult codons by optimizing the guideRNA that is applied in trans. We find that all 5′-XAG codons with X = U, A, C, G are editable in vitro to a substantial amount of at least 50% once the guideRNA/mRNA duplex is optimized. Notably, some codons, including CAG and GAG, accept or even require the presence of 5′-mismatched neighboring base pairs. This was unexpected from the reported analysis of global editing preferences on large double-stranded RNA substrates. Furthermore, we report the usage of guanosine mismatching as a means to suppress unwanted off-site editing in proximity to targeted adenosine bases. Together, our findings are very important to achieve selective and efficient editing in difficult codon and sequence contexts.Nucleic Acids Research 04/2014; 42(10). DOI:10.1093/nar/gku272 · 9.11 Impact Factor