The Role of Arg165 Towards Base Flipping, Base Stabilization and Catalysis in M.HhaI

Shieh, Fa Kuen; Youngblood, Benjamin A.; Reich, Norbert O.

doi:10.1016/j.jmb.2006.07.030

Cited by 43 publications

(79 citation statements)

References 48 publications

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“…Phe-101 and Phe-102 in the M.HhaI cognate⅐ternary complex (27) are nearly isomorphous with Phe-731 and Phe-732 in the DNMT3A complex (supplemental Fig. S2A).…”

Section: Resultsmentioning

confidence: 96%

Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation

Holz-Schietinger

Matje

Harrison

et al. 2011

Journal of Biological Chemistry

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103

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Background:De novo DNA methyltransferase 3A is essential for gene regulation, however its regulation is poorly understood. Results: DNMT3A dimers are fully functional but eliminate methylation on clustered CpG sites (processive catalysis) that are observed with the wild-type homotetramers. Conclusion: Protein binding by DNMT3A or DNMT3L at the tetramer interface controls the catalytic properties of DNMT3A. Significance: Provides a structural mechanism for the regulation of methylation patterning.

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“…Phe-101 and Phe-102 in the M.HhaI cognate⅐ternary complex (27) are nearly isomorphous with Phe-731 and Phe-732 in the DNMT3A complex (supplemental Fig. S2A).…”

Section: Resultsmentioning

confidence: 96%

Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation

Holz-Schietinger

Matje

Harrison

et al. 2011

Journal of Biological Chemistry

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103

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“…Moreover, we have selected E119 (from motif VI-ENV), R160 and R162 (from motif VIII-RXR) by their homology with the catalytic residues found in DNA m 5 C methyltransferases that were experimentally confirmed in different systems (see Fig. 1; Klimasauskas et al 1994;O'Gara et al 1999;Sankpal and Rao 2002;Reither et al 2003;Gowher et al 2006;Shieh et al 2006;Shieh and Reich 2007). We have purified these variants, confirmed their correct folding, and studied their RNA methylation activity and RNA binding.…”

Section: Resultsmentioning

confidence: 99%

Human DNMT2 methylates tRNA^Asp molecules using a DNA methyltransferase-like catalytic mechanism

Jurkowski¹,

Meusburger²,

Phalke³

et al. 2008

RNA

164

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Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA Asp . We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNAAsp confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

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“…1). Inspection of the first base pair of the recognition sequence in the 1.96 Å cocrystal structure (16) reveals no protein interactions to the cytosine base whereas three can be seen to the guanosine base, two from Arg-240 via the major groove to guanosine O 6 and N 7 and one from Ile-86 via the minor groove to guanosine N 2 (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Coupling Sequence-specific Recognition to DNA Modification

Estabrook

Nguyen

Fera

et al. 2009

Journal of Biological Chemistry

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Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M.HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is ϳ28 Å away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.Sequence-specific modification of DNA is essential for nearly all forms of life and contributes to a myriad of biological processes including gene regulation, mismatch repair, host defense, DNA replication, and genetic imprinting. Methylation of cytosine and adenine bases is a key epigenetic process whereby phenotypic changes are inherited without altering the DNA sequence (1). The central role of the bacterial and mammalian S-adenosylmethionine (AdoMet) 2 -dependent DNA methyltransferases in virulence regulation and tumorigenesis, respectively, have led these enzymes to be validated targets for antibiotic and cancer therapies (2, 3). However, AdoMet-dependent enzymes catalyze diverse reactions, and the design of potent and selective DNA methyltransferase inhibitors is particularly challenging (4, 5). The design of drugs that bind outside the active site is a particularly attractive means of inhibition for enzymes with common cofactors like AdoMet because off-target inhibition often leads to toxicity (6). Unfortunately, robust methods to identify and characterize such critical binding sites distal from the active site have not been developed.DNA methyltransferases bind to a particular DNA sequence, stabilize the target base into an extrahelical position within the enzyme active site, and transfer the methyl moiety from AdoMet to the DNA (7). During this process, dramatic changes in the DNA structure such as bending, base flipping, or the intercalation of residues into the recognition sequence are often accompanied by large scale protein rearrangements (8).Here we characterized a specific conformational rearrangement of M.HhaI, a model DNA cytosine C 5 methyltransferase with a cognate recognition sequence of 5Ј-GCGC-3Ј. Many structures of M.HhaI are available at high resolution including an ensemble of complexes with either cognate or nonspecific DNA (9, 10). Reorganization of an essential catalytic loop (residues 80 -100) is regulated by sequence-specific protein-DNA interactions that occur ϳ28 Å away from the catalytic loop (Fig. 1). Our work quantifies the importance of such distal communication in sequence-specific DNA modification and provides plausible structural mechanisms.DNA-dependent positioning of the catalytic loop in M.HhaI was first observed crystallographically; cognate DNA stabilizes the loop-closed conformer, while nonspecific DNA leaves the loop in the open c...

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The Role of Arg165 Towards Base Flipping, Base Stabilization and Catalysis in M.HhaI

Cited by 43 publications

References 48 publications

Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation

Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation

Human DNMT2 methylates tRNA^Asp molecules using a DNA methyltransferase-like catalytic mechanism

Coupling Sequence-specific Recognition to DNA Modification

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The Role of Arg165 Towards Base Flipping, Base Stabilization and Catalysis in M.HhaI

Cited by 43 publications

References 48 publications

Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation

Oligomerization of DNMT3A Controls the Mechanism of de Novo DNA Methylation

Human DNMT2 methylates tRNAAsp molecules using a DNA methyltransferase-like catalytic mechanism

Coupling Sequence-specific Recognition to DNA Modification

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Product

Resources

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Human DNMT2 methylates tRNA^Asp molecules using a DNA methyltransferase-like catalytic mechanism