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Query: EC:2.1.1.113 (restriction-modification system)
 350 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Survival and mutagenesis caused by 5-azacytidine was studied in Escherichia coli. Survival was partially lexA- and recA-dependent and was decreased by the presence of a DNA (cytosine-5)methyltransferase. The dcm, MspI, and EcoRII methyltransferase genes all decreased survival. There was no direct relationship between amount of methylase enzyme present and cell survival, but only plasmids containing a methylase gene sensitized cells to 5-azacytidine. Survival was not affected by uvrA, uvrB or umuCD mutations. Induction of sulA::lacZ fusions by 5-azacytidine was inhibited in strains containing elevated levels of DNA methylase. Cells resistant to 5-azacytidine when they contained a plasmid specifying the EcoRII methylase were sensitive if the plasmid specified the complete EcoRII restriction-modification system. The mechanism of cell death in these situations is therefore different. Mutation of the rpoB gene by 5-azacytidine was studied. The mutation rate was decreased by the presence of recA and lexA mutations. Mutation in umuCD had little effect on the mutation rate. The recA430 mutation, which does not support SOS-dependent mutagenesis induced by UV light, does support 5-azacytidine induced mutagenesis. The presence of DNA (cytosine-5)methyltransferase had no effect on the mutation rate caused by 5-azacytidine treatment. The mutagenic and lethal lesions caused by 5-azacytidine in the absence of methylase therefore differ from the lethal lesions that occur in the presence of methylase. The former could be due to the opening of the 5-azacytosine ring in DNA. Cell death in the presence of methylase could be due to tight binding of methylase to azacytosine containing DNA as well as inhibition of induction of the SOS response.
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PMID:Survival and mutagenic effects of 5-azacytidine in Escherichia coli. 245 47

The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R.FokI) and methyltransferase (M.FokI) are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M.FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a 'tandem' structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M. FokI were constructed by deleting the 5'- or 3'-ends of the fokIM gene. Deleting most of the C-terminus of M.FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M.FokI for methylating the two strands of the recognition sequence are largely separate.
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PMID:Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase. 268 65

The genes coding for the class-II Serratia marcescens restriction-modification system have been cloned and expressed in E. coli. Recombinant clones, restricted incoming phage only poorly; the recombinant plasmids, however, became fully modified in vivo, i.e. completely resistant against digestion with R.SmaI. The determined nucleotide sequence of the cloned system revealed three open reading frames with lengths of 252 bp, 741 bp, and 876 bp. Through various deletion experiments and an insertion-mutation experiment the 876 bp open reading frame could be assigned to the SmaI DNA modification enzyme and the 741 bp open reading frame to the SmaI restriction endonuclease. Mapping of the transcription start sites of the genes revealed that the SmaI endonuclease is transcribed as polycistronic mRNA together with a 252 bp long preceding open reading frame of unknown function. No homology was found when comparing the amino acid sequence of M.SmaI with the published sequences of m5C-specific DNA modification methyltransferases. On the other hand, a stretch of 14 amino acids in the C-proximal region of M.SmaI shows a significant homology to the C-proximal amino acid sequences of the N6A-methyltransferases M.HinfI and M.DpnIIA and the N4C-methyltransferase M.PvuII.
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PMID:Cloning, characterization and heterologous expression of the SmaI restriction-modification system. 269 8

An unusual cluster of tandemly repeated DNA sequences (TRS) was found downstream from the gene encoding DsaV methyltransferase, the DNA modification enzyme in the DsaV restriction-modification system found in a strain of Dactylococcopsis salina (Ds). The repeat unit is about 32-bp long and is present 13 times in the cluster. Each repeat unit can be divided into two distinct parts based on the level of sequence conservation and evolution. Hybridization of Ds DNA with a probe specific for this cluster revealed that there were at least two additional sites within the genome with similar TRS. The TRS units are localized in one region of the Ds genome. They do not share significant sequence similarity with other TRS found in prokaryotes.
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PMID:A novel repetitive sequence lies near the gene encoding a cytosine methyltransferase in the cyanobacterium Dactylococcopsis salina. 759 Mar 24

High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G. The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream. Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1. The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent.
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PMID:Complex transcription of an operon encoding the SalI restriction-modification system of Streptomyces albus G. 831 78

The DNA methyltransferase of the AluI restriction-modification system, from Arthrobacter luteus, converts cytosine to 5-methylcytosine in the sequence AGCT. The gene for this methyltransferase, aluIM, was cloned into Escherichia coli and sequenced. A 525-codon open reading frame was found, consistent with deletion evidence, and the deduced amino acid sequence revealed all ten conserved regions common to 5-methylcytosine methyltransferases. The aluIM sequence predicts a protein of M(r) 59.0k, in agreement with the observed M(r), making M.AluI the largest known methyltransferase from a type II restriction-modification system. M.AluI also contains the largest known variable region of any monospecific DNA methyltransferase, larger than that of most multispecific methyltransferases. In other DNA methyltransferases the variable region has been implicated as the sequence-specific target recognition domain. An in-frame deletion that removes a third of this putative target-recognition region leaves the Alu I methyltransferase still fully active.
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PMID:The M.AluI DNA-(cytosine C5)-methyltransferase has an unusually large, partially dispensable, variable region. 845 Nov 89

The contribution of nonspecific DNA to enzyme efficiency (k(cat)/K(m)) is described for a sequence-specific DNA-modifying enzyme. Our investigation focuses on the EcoRI DNA methyltransferase which transfers a methyl group from the cofactor S-adenosylmethionine to the second adenine in the double-stranded DNA sequence GAATTC. k(cat)/K(m) increases 4-fold as DNA length increases from 14 to 429 base pairs and increases 2-fold as the distance from the site to the nearest end is increased from 29 to 378 base pairs. No changes in k(cat)/K(m) result from further increases in either case. A facilitated diffusion mechanism is proposed in which the methyltransferase scans an average of <400 base pairs prior to dissociation from a DNA molecule. The methyltransferase was found to methylate two sites on a single DNA molecule in a distributive rather than a processive manner, suggesting that the enzyme dissociates from the DNA prior to release of the reaction product S-adenosylhomocysteine. A direct competition experiment with the EcoRI endonuclease shows the methyltransferase to be slightly more efficient at specific site location and catalysis. A rationale for the role of facilitated diffusion in this type II restriction-modification system is proposed.
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PMID:Contribution of facilitated diffusion and processive catalysis to enzyme efficiency: implications for the EcoRI restriction-modification system. 865 61

The salIR and salIM genes encode the endonuclease and methyltransferase components of the SalI restriction-modification system from Streptomyces albus G. Expression of the salI genes in Escherichia coli was investigated and major differences with Streptomyces were found. In E. coli there is no detectable expression of the salI R gene due to inactivity of the sal-pR promoter region. In the natural host of the system this region directs transcription of the salI genes as a bicistronic message. In contrast to salIR, salIM is transcribed in the heterologous host from a promoter within the salI DNA. Since sal-pR is not active, the gene cannot be expressed as part of the salI operon. It is probably transcribed from sal-pM, a promoter internal to the operon which allows independent expression of the modification gene in Streptomyces. Replacement of sal-pR by the strong pLac promoter allows expression of salIR in E. coli and enhances expression of salIM. The resulting strain produces about 10 times more endonuclease than a Streptomyces clone containing the SalI system under the control of sal-pR.
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PMID:Comparative analysis of expression of the SalI restriction-modification system in Escherichia coli and Streptomyces. 900 89

The gene (xamIM) encoding the DNA methyltransferase of the XamI restriction-modification system from Xanthomonas campestris pv. amaranithicola (M.XamI) has been cloned in Escherichia coli and its nucleotide sequence determined. The sequence predicts a protein of 527 amino acids that contains nine conserved motifs characteristic of DNA amino methyltransferases. In fact, M.XamI shows significant similarity with N6-adenine methyltransferases of the gamma group of amino methyltransferases, including M.SalI (from the isoschizomeric SalI restriction-modification system) and M.TaqI (the only N6-adenine methyltransferase for which a three-dimensional structure is available). M.XamI and M.SalI share two highly conserved regions within the C-terminal domain, one of which aligns with one of the DNA recognition loops proposed for M.TaqI. Analysis of the chromosomal DNA adjacent to xamIM led to the identification of an additional ORF (275 codons), downstream, in the same transcriptional orientation. Although some limited similarities between the SalI restriction enzyme and the product deduced from this ORF were found, the clone carrying xamIM did not express the expected endonuclease function.
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PMID:Isolation and nucleotide sequence of the gene encoding the XamI DNA methyltransferase of Xanthomonas campestris pv. amaranthicola. 913 May 89

The regulation of the Sso II restriction-modification system from Shigella sonnei was studied in vivo and in vitro . In lacZ fusion experiments, Sso II methyltransferase (M. Sso II) was found to repress its own synthesis but stimulate expression of the cognate restriction endonuclease (ENase). The N-terminal 72 amino acids of M. Sso II, predicted to form a helix-turn-helix (HTH) motif, was found to be responsible for the specific DNA-binding and regulatory function of M. Sso II. Similar HTH motifs are predicted in the N-terminus of a number of 5-methylcytosine methyltransferases, particularly M. Eco RII, M.dcm and M. Msp I, of which the ability to regulate autogenously has been proposed. In vitro, the binding of M. Sso II to its target DNA was investigated using a mobility shift assay. M. Sso II forms a specific and stable complex with a 140 bp DNA fragment containing the promoter region of Sso II R-M system. The dissociation constant (Kd) was determined to be 1.5x10(-8) M. DNaseI footprinting experiments demonstrated that M. Sso II protects a 48-52 bp region immediately upstream of the M. Sso II coding sequence which includes the predicted -10 promoter sequence of M. Sso II and the -10 and -35 sequences of R. Sso II.
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PMID:Specific binding of sso II DNA methyltransferase to its promoter region provides the regulation of sso II restriction-modification gene expression. 915 10


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