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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genes coding for the MunI restriction-modification (R-M) system, which recognize the sequence 5'-CAATTG, have been cloned and expressed in Escherichia coli, and their nucleotide sequences have been determined. The restriction endonuclease (ENase; R.MunI) is encoded by an open reading frame (ORF) of 606 bp, and a 699-bp ORF codes for the methyltransferase (MTase). The two genes are transcribed divergently from a 355-bp region. The gene encoding the ENase is preceded by a short co-linear ORF of 222 bp. The deduced amino acid (aa) sequence of this short ORF (SORF) closely resembles the sequences of a family of regulatory proteins that are associated with other type-II R-M systems. Comparative analysis of the deduced aa sequence of R.MunI revealed several regions of similarity to the EcoRI and RsrI ENases that recognize the GAATTC sequence. The similar mode of interaction of MunI, EcoRI and RsrI with the tetranucleotide AATT, common to the recognition sequences of these ENases, was suggested.
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PMID:CAATTG-specific restriction-modification munI genes from Mycoplasma: sequence similarities between R.MunI and R.EcoRI. 818 41

Two different clonal groups of pathogenic Yersinia enterocolitica strains, American and non-American, have been recognized. These are distinguished by a number of criteria, including their virulence in a murine model of infection. However, genetic analysis of virulence in American strains has been hampered due to the severe restriction of transformed or electroporated DNA. Thus, we cloned the yenIMR locus from the American serotype strain 8081c, which encodes YenI, an isoschizomer of PstI. This clone encodes both the restriction endonuclease and methyltransferase. The location of the genes on the clone was determined and this information was used to construct a small deletion (400 bp) that results in an R-M+ phenotype. This mutation was recombined onto the Y. enterocolitica chromosome to give an R-M+ mutant which showed at least a 1000-fold increase in electroporation frequency compared to the wild-type strain. Southern analysis using a probe derived from yenIMR indicated that American serotype strains have this locus whereas non-American serotype strains do not.
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PMID:Cloning of the YenI restriction endonuclease and methyltransferase from Yersinia enterocolitica serotype O8 and construction of a transformable R-M+ mutant. 829 16

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 genes encoding the CfrBI restriction and modification (R-M) systems from Citrobacter freundii and recognizing the sequence 5'-CCWWGG-3' (W = A or T) were cloned in Escherichia coli McrBC- cells. The nucleotide (nt) sequences of the genes were determined. Two large open reading frames were found. Deletion analysis showed that one of them [1128 nt coding for 376 amino acids (aa)] corresponds to a methyltransferase (MTase)-encoding gene and the other (1065 nt coding for 355 aa) to a restriction endonuclease-encoding gene. The genes are oriented divergently and separated by 76 bp. A CfrBI site (5'-m4CCATGG) was found in the intergenic region of the cfrBIRM genes. Analysis of the deduced aa sequence of M.CfrBI made it possible to determine the typical features of a m4C-specific MTase. Limited homology between the M.CfrBI and R.CfrBI proteins was also found.
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PMID:Cloning and sequences of the genes encoding the CfrBI restriction-modification system from Citrobacter freundii. 833 62

The restriction endonuclease Cac824I has been shown to be a major barrier to electrotransformation of Clostridium acetobutylicum ATCC 824 (L. D. Mermelstein, N. E. Welker, G. N. Bennett, and E. T. Papoutsakis, Bio/Technology 10:190-195, 1992). Methylation by the phi 3T I methyltransferase encoded by Bacillus subtilis phage phi 3T was shown to protect plasmid DNA from restriction by Cac824I. Expression in Escherichia coli of the phi 3tI gene (which encodes the phi 3T I methyltransferase) from pAN1, which replicates via the p15A origin of replication, was sufficient to completely methylate coresident E. coli-C. acetobutylicum shuttle vectors with ColE1 origins of replication. Three shuttle vectors (pIMP1, pSYL2, and pSYL7) methylated in this manner were used to efficiently electrotransform strain ATCC 824. These vectors could not be introduced into strain ATCC 824 when unmethylated because the E. coli portions of the plasmids contain a large number of Cac824I sites. This method obviates the need to use B. subtilis-C. acetobutylicum shuttle vectors with few Cac824I sites to introduce DNA into C. acetobutylicum ATCC 824.
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PMID:In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. 838

The NgoII restriction-modification (R-M) system of Neisseria gonorrhoeae recognizes the sequence 5'-GGCC-3'. This system is encoded by two separate genes, dcmB for the methyltransferase (MTase) and dcrB for the restriction endonuclease (ENase). Three strains that vary in their NgoII phenotype were examined. Strain Pgh3-2 produced detectable levels of both enzymes, strain F62 lacked detectable levels of the dcrB gene product, and strain WR302 failed to produce either gene product. Strains that lacked either enzyme activity still possessed the genes that encode them. Transcriptional fusions of dcrB in strains F62 and Pgh3-2 indicate that this gene is transcribed at nearly identical levels in each strain. The DNA encoding the NgoII R-M system was cloned from the three strains, and the nucleotide sequence was determined. The dcrB genes of WR302 and F62 possess the same frameshift mutation (base position 1435) which would result in a truncated protein. The WR302 dcmB was found to have a point mutation that changed Arg288 (a residue that is conserved in all prokaryotic and phage cytosine MTases sequenced to date) to Trp.
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PMID:Natural variation of the NgoII restriction-modification system of Neisseria gonorrhoeae. 840 39

The genes hsdM and hsdS for M. EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R. EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42 degrees C. In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutant hsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of the hsdSts-1 mutation on restriction and modification.
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PMID:Overproduction of the Hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype. 854 91

The EcoRV methyltransferase modifies DNA by the introduction of a methyl group at the 6-NH2 position of the first deoxyadenosine in GATATC sequences. The enzyme forms a stable and specific complex with GATATC sequences in the presence of a nonreactive analogue, such as sinefungin, of its natural cofactor S-adenosyl-L-methionine. Using circular permutation band mobility shift analysis (in which the distance between the GATATC sequence and the end of the DNA is varied) of protein-DNA-cofactor complexes we have shown the methylase induces a bend of just over 60 degrees in the bound DNA. This was confirmed by phasing analysis, in which the spacing between the GATATC site and a poly(dA) tract is varied through a helical turn, which showed that the orientation of the induced curve was toward the major groove. There was no significant difference in the bend angle measured using unmethylated GATATC sequences and hemimethylated sequences which contain G6-Me ATATC in one strand only. These are the natural substates for the enzyme. The EcoRV endonuclease, a very well characterized protein, served as a positive control. DNA bending by this protein has been previously determined both by crystallographic and solution methods. The two proteins bend DNA toward the major groove but the bend angle produced by the methylase, slightly greater than 60 degree, is a little larger than that observed with the endonuclease, which is approximately 44 degrees.
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PMID:The EcoRV modification methylase causes considerable bending of DNA upon binding to its recognition sequence GATATC. 855 24

Two segments of the gene for the EcoRV restriction endonuclease, each encoding 10 amino acids at the active site, were subjected to random mutagenesis with degenerate oligonucleotides. Mutations that abolished the activity of the EcoRV endonuclease were selected by viability in a strain of Escherichia coli that lacks the EcoRV methyltransferase, under conditions where the gene for the wild-type endonuclease is lethal to the cell. Sixty-five mutants were isolated and analyzed by DNA sequencing to identify the mutations. The collection of null mutants contained 49 with single amino acid substitutions, 15 with double substitutions, and one with a triple substitution. The single substitutions were located at many different positions within the two 10-amino acid segments, though several hot-spots gave rise to null mutants at high frequencies. Some hot-spots were readily explained by reference to the crystal structure of EcoRV since they were at the amino acids immediately adjacent to the scissile phosphodiester bond: for example, Asp90 and Lys92. These residues may be directly involved in the catalytic mechanism. Other hot-spots, such as Gln69, Tyr72, and Ala88, were at unexpected positions that appear to have no direct role in DNA binding or catalysis. At some of the unexpected hot-spots, the side chain of the amino acid lies distant from the DNA, yet the enzyme was still inactivated by conservative substitutions at these positions. The sensitivity of the EcoRV endonuclease to conservative substitutions may be due to its requirement to take up one particular conformation at the DNA-protein interface out of a large number of alternative conformations.
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PMID:Random mutagenesis targeted to the active site of the EcoRV restriction endonuclease. 863 49

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


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