Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.1.1.37 (DNA methyltransferase)
 4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence d(N-G-A-A-T-T-C-N)/d(N-C-T-T-A-A-G-N) when the ionic strength is high, 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 mM Tris-HCl, 2 mM MgCl2, and adjusting the pH to 8.5 reduces the recognition specificity of the EcoRI endonuclease to the tetranucleotide sequence, d(N-A-A-T-T-N)/d(N-T-T-A-A-N). The enzymatic activity responsible for this substrate recognition is referred to as EcoRI. Cleavage of pVH51 plasmid DNA under EcoRI conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites are cleaved at different rates. Comparison of DNA fragment patterns of modified and unmodified pVH51 DNA indicates that the canonical EcoRI sequence is the most rapidly cleaved site under EcoRI conditions. DNA modified in vivo by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under EcoRI conditions at sites other than the standard EcoRI substrate.
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PMID:Specificity of substrate recognition by the EcoRI restriction endonuclease. 24 1

(Deoxyribonucleic acid from Micrococcus luteus was methylated in vitro in the presence of S-adenosyl-(14C methyl)methionine with a DNA methyltransferase purified from extracts of te. coli infected with bacteriophage T2. The labelled DNA was degraded by enzymatic and specific chemical methods and the resulting short oligonucleotides were separated and characterized. tthe analytical data permit the conclusion that the tdna transmethylase reacts specifically with N-G-A-T-C-N sequences in which it converts adenine to a 6-methyl-aminopurine residue.
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PMID:Specificity of deoxyribonucleic acid transmethylase induced by bacteriophage T2. I. Nucleotide sequences isolated from tmicrococcus luteus DNA methylated in vitro. 110 Dec 23

The structural gene (dpnM) for the Dpn II DNA methylase of Streptococcus pneumoniae, which is part of the Dpn II restriction system and methylates adenine in the sequence 5'-G-A-T-C-3', was identified by subcloning fragments of a chromosomal segment from a Dpn II-producing strain in an S. pneumoniae host/vector cloning system and demonstrating function of the gene also in Bacillus subtilis. Determination of the nucleotide sequence of the gene and adjacent DNA indicates that it encodes a polypeptide of 32,903 daltons. A putative promoter for transcription of the gene lies within a hundred nucleotides of the polypeptide start codon. Comparison of the coding sequence to that of the dam gene of Escherichia coli, which encodes a similar methylase, revealed 30% of the amino acid residues in the two enzymes to be identical. This homology presumably reflects a common origin of the two genes prior to the divergence of Gram-positive and Gram-negative bacteria. It is suggested that the restriction function of the gene is primitive, and that the homologous restriction system in E. coli has evolved to play an accessory role in heteroduplex DNA base mismatch repair.
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PMID:Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli. 298 23

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.
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PMID:The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences. 301 80

We have examined the DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues 2-aminopurine, 5-bromocytosine, 5-bromouracil, 2,6-diaminopurine, hypoxanthine, 5-methylcytosine, N6-methyladenine, and uracil were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the ability of the enzyme to methylate the modified substrates were monitored by determining the steady state kinetic values of the reaction in the presence of the cosubstrate, S-adenosylmethionine. The substitutions resulted in effects ranging from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those analogues that were active, whereas the octanucleotides containing hypoxanthine at the guanine site, N6-methyladenine at the first or 2-aminopurine at the second adenine site, or uracil at the second thymine site were completely inactive. The results are discussed in terms of the possible interactions between the methylase and its recognition sequence. In addition, the interactions are compared to those of the EcoRI restriction endonuclease, which has been similarly tested with the same analogue oligonucleotides. The results of that study are reported in an accompanying paper. Although both enzymes recognize the same sequence, they do so in different ways.
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PMID:The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences. 301 81

The DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) has increasingly attracted worldwide attention for its antineoplastic potential. The cytotoxitic mechanisms, however, especially, the relative contribution of silenced genes reactivation by demethylation and enzyme-DNA adduct formation to the efficacy of 5-Aza-CdR is still a crucial unresolved question. In this investigation, we demonstrated that 5-Aza-CdR treatment resulted in growth suppression in a concentration and time-dependent manner and G2 phrase arrest - hallmarks of a DNA damage response in gastric cancer AGS cells. Formation of DNA double-strand breaks, as monitored by comet assay was examined in an ATM (ataxia-telangiectasia mutated)-dependent manner based on the fact that PI3K inhibitor Wortmannin abolished the action of cytotoxicity of 5-Aza-CdR. Upon treatment with 5-Aza-CdR, ATM activation was clearly associated with P53 phosphorylation at Ser(15), which was directly responsible for 5-Aza-CdR modified P21(Waf1/Cip1) expression. Further exploration revealed that demethylation of P16(INK4A) correlated with the strikingly down-regulated expressions of DNA methyltransferase 3A as well as 3B was, at least in part, attributed to the cytotoxicity of 5-Aza-CdR in AGS cells. Conclusively, these results greatly enhance our understanding of the mechanisms of cytotoxicity of 5-Aza-CdR and strongly provide the preclinical rationale for an assessment of 5-Aza-CdR to ameliorate patient outcome with gastric cancer.
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PMID:Cytotoxicity of 5-Aza-2'-deoxycytidine against gastric cancer involves DNA damage in an ATM-P53 dependent signaling pathway and demethylation of P16(INK4A). 2320 Oct 8