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 arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity. None of the mutant enzymes show a preference for alternative sites in assays in vitro. The implications of these results are discussed.
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PMID:Determinants of EcoRI endonuclease sequence discrimination. 265 23

The Eco RI endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467). The amino acid sequences of both enzymes were derived from the DNA sequence. The coding regions selected include the only open translational frames of sufficient length to accommodate the enzymes. They coincide with previously established gene boundaries and orientation. The predicted amino acid sequences correlate well with analyses of the purified protein. Comparison of the nucleotide and protein sequences reveals no homology between the endonuclease and methylase which might provide insight into the origin of the restriction-modification system or the mechanism of common substrate recognition. Based on secondary structure predictions, the two enzymes also have grossly different molecular architecture. The base composition of the sequence is 65% A + T, and the codon usage is significantly different from that observed in several Escherichia coli chromosomal genes. In some cases, frequently selected codons are recognized by minor tRNA species. A spontaneous mutation in the endonuclease gene was isolated. Serine replaces arginine at residue 187. In crude extracts, Eco RI specific cleavage is approximately 0.3% wild type.
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PMID:Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase. 625 3

The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.
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PMID:Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription. 786 1

O6-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.
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PMID:Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening. 820 83

The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG). The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site. One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases. The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E. coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4 degrees C and 37 degrees C. The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37 degrees C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4 degrees C. Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures. Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold. We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.
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PMID:The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity. 836 18

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue.
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PMID:Functional analysis of conserved motifs in EcoP15I DNA methyltransferase. 865 25

O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, acts as a monomer in removing the mutagenic DNA adduct O6-alkylguanine (induced by alkylating carcinogens) via a stoichiometric reaction. The alkyl group is transferred without a cofactor to a specific cysteine acceptor residue of MGMT, Cys-145 in the case of human MGMT, containing 207 amino acid residues and thereby inactivates the protein. As a prelude to the investigation of the reaction mechanism of human MGMT by elucidation of its structure in free and substrate-bound forms via NMR spectroscopy and X-ray crystallography, two types of MGMT mutants were generated and characterized. First, systematic deletion analysis of the protein was carried out to determine the smallest size at which it is active or inactive but forms a stable complex with the substrate and so may be useful for NMR spetroscopic analysis. Deletion of more than 8 or 31 residues from the amino or carboxyl terminus, respectively, led to the loss of both activity and substrate binding. Removal of Arg-9 or Leu-176 and distal residues inactivated the protein, presumably by altering its tertiary structure. On the basis of the criteria of bacterial overexpression and solubility, the mutant MGMT with deletion of 28 residues at the carboxyl terminus should be suitable for NMR studies. In the second approach, we examined mutants at the active site (Cys-145) that retain substrate binding. Inactive C145A and C145S substitution mutants were found to form specific and stable complexes with an O6-methylguanine (m6G)-containing oligonucleotide substrate. Wild type MGMT also formed a similar complex, but only as a transient intermediate. Footprinting studies indicated a strong discriminatory effect of the base adduct on the binding of C145A to substrate DNA; 17-18 nucleotides on the m6G-containing strand and 13-14 nucleotides in the complementary strand spanning the base adduct were protected from DNase I digestion by the mutant protein. These results, as well as the identical protease sensitivity of the wild type and mutant proteins, suggest minimal structural change due to conservative mutations at the active site. Thus, the mutant proteins may be utilized for solving the structure and mechanism of human MGMT.
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PMID:Specific recognition of O6-methylguanine in DNA by active site mutants of human O6-methylguanine-DNA methyltransferase. 915 17

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.
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PMID:A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway. 1109 Feb 75

DNA methylation is involved in epigenetic processes such as X-chromosome inactivation, imprinting and silencing of transposons. We have demonstrated previously that dim-2 encodes a DNA methyltransferase that is responsible for all known cytosine methylation in Neurospora crassa. Here we report that another Neurospora gene, dim-5, is required for DNA methylation, as well as for normal growth and full fertility. We mapped dim-5 and identified it by transformation with a candidate gene. The mutant has a nonsense mutation in a SET domain of a gene related to histone methyltransferases that are involved in heterochromatin formation in other organisms. Transformation of a wild-type strain with a segment of dim-5 reactivated a silenced hph gene, apparently by 'quelling' of dim-5. We demonstrate that recombinant DIM-5 protein specifically methylates histone H3 and that replacement of lysine 9 in histone H3 with either a leucine or an arginine phenocopies the dim-5 mutation. We conclude that DNA methylation depends on histone methylation.
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PMID:A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. 1171 9

Chloroplast DNA of the green alga Chlamydomonas reinhardtii is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt(+)) gametes is heavily methylated whereas that of paternal (mating type minus; mt(-)) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt(+) gametes, and not in mt(-) gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses. CrMET1 transcripts were found to be absent in both mt(+) and mt(-) vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt(+) but not mt(-) cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt(+) gametes. This conclusion was further strengthened by the observation that, during gametogenesis, CrMET1 is expressed in a mt(-) mutant, mat-1, whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in C. reinhardtii.
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PMID:A chloroplast-resident DNA methyltransferase is responsible for hypermethylation of chloroplast genes in Chlamydomonas maternal gametes. 1198 92


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