Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The EcoRV DNA methyltransferase introduces a CH3 group at the 6-amino position of the first dA in the duplex sequence d(GATATC). It has previously been reported that the methylase contacts the four phosphates (pNpNpGpA) at, and preceding, the 5'-end of the recognition sequence as well as the single dG in this sequence (Szczelkun, M. D., Jones, H., and Connolly, B. A. (1995) Biochemistry 34, 10734-10743). To study the possible role of the dA and T bases within the ATAT sequence, interference studies have been carried out using diethylpyrocarbonate and osmium tetroxide. The methylase bound very strongly to hemimethylated oligonucleotides modified at the second AT, of the ATAT sequence, in the unmethylated strand of the duplex. This probably arises because these modifications facilitate DNA distortion that follows the binding of the nucleic acid to the protein. Oligonucleotides containing modified bases at both the target dA base and its complementary T were used to determine whether this dA methylase flips out its target base in a similar manner to that observed for dC DNA methylases. In binary EcoRV methylase-DNA complexes, analogues that weakened the base pair caused an increase in affinity between the protein and the nucleic acid. In contrast, in ternary EcoRV methylase-DNA-sinefungin (an analogue of the natural co-factor, S-adenosyl-L-methionine (AdoMet)) complexes, only small differences in affinity were observed between the normal dA-T base pair and the analogues. These results are almost identical to those seen with DNA dC methylases (Klimasauskas, S., and Roberts R. J. (1995) Nucleic Acid Res. 23, 1388-1395; Yang, S. A., Jiang-Cheng, S., Zingg, J. M., Mi, S., and Jones, P. A. (1995) Nucleic Acids Res. 23, 1380-1387) and support a base-flipping mechanism for DNA dA methylases.
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PMID:DNA distortion and base flipping by the EcoRV DNA methyltransferase. A study using interference at dA and T bases and modified deoxynucleosides. 899 88

The crystal structures of the binary complexes of the DNA methyltransferase M.TaqI with the inhibitor Sinefungin and the reaction product S-adenosyl-L-homocysteine were determined, both at 2.6 A resolution. Structural comparison of these binary complexes with the complex formed by M.TaqI and the cofactor S-adenosyl-L-methionine suggests that the key element for molecular recognition of these ligands is the binding of their adenosine part in a pocket, and discrimination between cofactor, reaction product and inhibitor is mediated by different conformations of these molecules; the methionine part of S-adenosyl-L-methionine is located in the binding cleft, whereas the amino acid moieties of Sinefungin and S-adenosyl-L-homocysteine are in a different orientation and interact with the active site amino acid residues 105NPPY108. Dissociation constants for the complexes of M.TaqI with the three ligands were determined spectrofluorometrically. Sinefungin binds more strongly than S-adenosyl-L-homocysteine or S-adenosyl-L-methionine, with KD=0.34 microM, 2.4 microM and 2.0 microM, respectively.
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PMID:Differential binding of S-adenosylmethionine S-adenosylhomocysteine and Sinefungin to the adenine-specific DNA methyltransferase M.TaqI. 899 24

The murine DNA methyltransferase catalyzes the transfer of methyl groups from S-adenosylmethionine to cytosines within d(CpG) dinucleotides. The enzyme is necessary for normal embryonic development and is implicated in a number of important processes, including the control of gene expression and cancer. Metabolic labeling and high pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) were performed on DNA methyltransferase purified from murine erythroleukemia cells. Serine 514 was identified as a major phosphorylation site that lies in a domain required for targeting of the enzyme to the replication foci. These results present a potential mechanism for the regulation of DNA methylation. HPLC-ESI-MS peptide mapping data demonstrated that the purified murine DNA methyltransferase protein contains the N-terminal regions predicted by the recently revised 5' gene sequences (Yoder, J. A., Yen, R.-W. C., Vertino, P. M., Bestor, T. H. , and Baylin, S. B. (1996) J. Biol. Chem. 271, 31092-31097). The evidence suggests a start of translation at the first predicted methionine, with no alternate translational start sites. Our peptide mapping results provide a more detailed structural characterization of the DNA methyltransferase that will facilitate future structure/function studies.
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PMID:Peptide mapping of the murine DNA methyltransferase reveals a major phosphorylation site and the start of translation. 921 41

The BcgI restriction-modification system consists of two subunits, A and B. It is a bifunctional protein complex which can cleave or methylate DNA. The regulation of these competing activities is determined by the DNA substrates and cofactors. BcgI is an active endonuclease and a poor methyltransferase on unmodified DNA substrates. In contrast, BcgI is an active methyltransferase and an inactive endonuclease on hemimethylated DNA substrates. The cleavage and methylation reactions share cofactors. While BcgI requires Mg2+and S -adenosyl methionine (AdoMet) for DNA cleavage, its methylation reaction requires only AdoMet and yet is significantly stimulated by Mg2+. Site-directed mutagenesis was carried out to investigate the relationship between AdoMet binding and BcgI DNA cleavage/methylation activities. Most substitutions of conserved residues forming the AdoMet binding pocket in the A subunit abolished both methylation and cleavage activities, indicating that AdoMet binding is an early common step required for both cleavage and methylation. However, one mutation (Y439A) abolished only the methylation activity, not the DNA cleavage activity. This mutant protein was purified and its methylation, cleavage and AdoMet binding activities were tested in vitro . BcgI-Y439A had no detectable methylation activity, but it retained 40% of the AdoMet binding and DNA cleavage activities.
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PMID:Substrate DNA and cofactor regulate the activities of a multi-functional restriction-modification enzyme, BcgI. 927 91

EcoP1I and EcoP15I are members of type III restriction-modification enzymes. EcoPI and EcoP15I DNA methyltransferases transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the second adenine residues in their recognition sequences, 5'-AGACC-3' and 5'-CAGCAG-3' respectively. We have altered various residues in two highly conserved sequences, FxGxG (motif I) and DPPY (motif IV) in these proteins by site-directed mutagenesis. Using a mixture of in vivo and in vitro assays, our results on the mutational analysis of these methyltransferases demonstrate the universal role of motif I in AdoMet binding and a role for motif IV in catalysis. All six cysteine residues in EcoP15I DNA methyltransferase have been substituted with serine and the role of cysteine residues in EcoP15I DNA methyltransferase catalysed reaction assessed. The Res subunits of type III restriction enzymes share a distant sequence similarity with and contain the motifs characteristic of the DEAD box proteins. We have carried out site-directed mutagenesis of the conserved residues in two of the helicase motifs of the EcoP1I restriction enzyme in order to investigate the role of motifs in DNA cleavage by this enzyme. Our findings indicate that certain conserved residues in these motifs are involved in ATP hydrolysis while the other residues are involved in coupling restriction of DNA to ATP hydrolysis. Taken collectively, these results form the basis for a detailed structure-function analysis of EcoP1I and EcoP15I restriction enzymes.
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PMID:Functional analysis of conserved motifs in type III restriction-modification enzymes. 962 45

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.
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PMID:Probing the role of cysteine residues in the EcoP15I DNA methyltransferase. 972 99

DNA (cytosine-5-)-methyltransferase is essential for viable mammalian development and has a central function in the determination and maintenance of epigenetic methylation patterns. Steady-state and substrate trapping studies were performed to better understand how the enzyme functions. The catalytic efficiency was dependent on substrate DNA length. A 14-fold increase in KmDNA was observed as the length decreased from 5000 to 100 base pairs and kcat decreased by a third. Steady-state analyses were used to identify the order of substrate addition onto the enzyme and the order of product release. Double-reciprocal patterns of velocity versus substrate concentration intersected far from the origin and were nearly parallel. The kinetic mechanism does not appear to change when the DNA substrate is either 6250 or 100 base pairs in length. Isotope trapping studies showed that the initial enzyme-AdoMet complex was not catalytically competent; however, the initial enzyme-poly(dI.dC-dI.dC) complex was observed to be competent for catalysis. Product inhibition studies also support a sequential ordered bi-bi kinetic mechanism in which DNA binds to the enzyme first, followed by S-adenosyl-L-methionine, and then the products S-adenosyl-L-homocysteine and methylated DNA are released. The proposed mechanism is similar to the mechanism proposed for M. HhaI, a bacterial DNA (cytosine-5-)-methyltransferase. Evidence for an enzyme-DNA-DNA ternary complex is also presented.
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PMID:Murine DNA (cytosine-5-)-methyltransferase: steady-state and substrate trapping analyses of the kinetic mechanism. 979 Jun 80

The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.
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PMID:Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus. 985 52

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.
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PMID:Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus. 993 Oct 7

Structural studies of the proteins of the BstVI restriction-modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 degrees C and 55 or 60 degrees C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3-5 microM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 microM at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 degrees C and 55 degrees C. These results were interpreted in terms of differences in the structural characteristics of these restriction-modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 degrees C and at the temperature at which they are most active.
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PMID:Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy. 1042 88


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