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)

A plasmid (5.4 kbp) from Salmonella Typhi D4 has been identified as encoding a restriction and modification (R-M) system. DNA fragments (2537 bp) that carried the genes for restriction endonuclease and methyltransferase encoded on the plasmid were sequenced. Two divergently arranged open reading frames of 957 bp for the restriction endonuclease consisting of 318 aa (amino acids) and 1140 bp for the DNA methyltransferase consisting of 379 aa were identified. These sequences were similar to the sequences of the SsoII R-M system, including the interspace between the two genes.
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PMID:StyD4I restriction-modification system of Salmonella typhi D4: cloning and sequence analysis. 905 87

Bg/II, a type II restriction-modification (R-M) system from Bacillus globigii, recognizes the sequence 5'-AGATCT-3'. The system has been cloned into E. coli in multiple steps: first the methyltransferase (MTase) gene, bglIIM, was cloned from B. globigii RUB561, a variant containing an inactivated endonuclease (ENase) gene (bglIIR). Next the ENase protein (R.BglII) was purified to homogeneity from RUB562, a strain expressing the complete R-M system. Oligonucleotide probes specific for the 5' end of the gene were then synthesized and used to locate bglIIR, and the gene was isolated and cloned in a subsequent step. The nucleotide sequence of the system has been determined, and several interesting features have been found. The genes are tandemly arranged, with bglIIR preceding bglIIM. The amino acid sequence of M.BglII is compared to those of other known MTases. A third gene encoding a protein with sequence similarity to known C elements of other R-M systems is found upstream of bglIIR. This is the first instance of a C gene being associated with an R-M system where the R and M genes are collinear. In addition, open reading frames (ORFs) resembling genes involved with DNA mobility are found in close association with BglII. These may shed light on the evolution of the R-M system.
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PMID:Cloning and characterization of the Bg/II restriction-modification system reveals a possible evolutionary footprint. 907 62

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

The nucleotide sequence of the chromosomally encoded type II ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503 was completed. The ScrFI restriction endonuclease (ENase) has previously been shown to specifically recognize 5' CCNGG 3' sites, cleaving after the second cytosine and the degenerate central base. The ENase gene (scrFIR; 362 bp) was located between, and co-directionally transcribed with, two formerly characterized 5-methylcytosine methyltransferase genes, which encodes proteins that independently confer protection against ScrFI digestion. scrFIR codes for a protein of 272 amino acids with a predicted molecular mass of 31470 Da, which agrees favourably with a previously estimated molecular mass of 34 kDa for this enzymes. The deduced sequence of this protein did not show any significant homology with known protein sequences, including the isoschizomeric Ssoll ENase from Shigella sonnei. The ENase gene was cloned and expressed in Escherichia coli and Lactococcus; however, no in vivo restriction of phage was observed, suggesting that expression of the ENase gene may be repressed, or that the appropriate expression signals may be absent in the cloned constructs. The ability of ScrFI to cleave non-canonically modified 5' CCNGG 3' sequences suggested that some ScrFI sites may require complex modifications to fully impair digestion by this enzyme.
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PMID:Molecular characterization of the restriction endonuclease gene (scrFIR) associated with the ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503. 924 16

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

Two genes from Corynebacterium equii, a Gram-positive bacterium producing the CeqI restriction-modification enzymes were cloned and sequenced. In vivo restriction experiments, DNA and amino acid sequence data suggest that the two genes code for the endonuclease and the methyltransferase enzymes. However, when the two genes are expressed in E. coli, practically no enzyme activity can be detected in the supernatants of sonicated cells. Based on the DNA sequence data CeqI restriction endonuclease (an EcoRV izoschizomer) consists of 270 amino acid residues with a predicted molecular mass of 31.6 kDa, in good agreement with the previously measured 32 +/- 2 kDa. The methyltransferase is 517 residues long (approx. 60 kDa). The two genes are in opposite orientation and overlap by 37 base pairs on the chromosome. The deduced amino acid sequence of the putative endonuclease gene revealed long stretches of hydrophobic amino acids, that may form the structural basis of the unusual aggregation properties of the restriction endonuclease. The amino acid sequence of the methylase shows homologies with other type II methyltransferases.
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PMID:Cloning and characterization of the genes of the CeqI restriction-modification system. 930 4

BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector.
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PMID:Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs. 932 48

Bcg I and Bcg I-like restriction endonucleases cleave double stranded DNA specifically on both sides of their asymmetric recognition sequences which are interrupted by several ambiguous base pairs. Their heterosubunit structure, bifunctionality and stimulation by AdoMet make them different from other classified restriction enzymes. Here we report on a new Bcg I-like restriction endonuclease, Bpl I from Bacillus pumilus , which in contrast to all other Bcg I-like enzymes, recognizes a symmetric interrupted sequence, and which, like Bcg I, cleaves double stranded DNA upstream and downstream of its recognition sequence (8/13)GAGN5CTC(13/8). Like Bcg I, Bpl I is a bifunctional enzyme revealing both DNA cleavage and methyltransferase activities. There are two polypeptides in the homogeneous preparation of Bpl I with molecular masses of approximately 74 and 37 kDa. The sizes of the Bpl I subunits are close to those of Bcg I, but the proportion 1:1 in the final preparation is different from that of 2:1 in Bcg I. Low activity observed with Mg2+increases >100-fold in the presence of AdoMet. Even with AdoMet though, specific cleavage is incomplete. S -adenosylhomocysteine (AdoHcy) or sinefungin can replace AdoMet in the cleavage reaction. AdoHcy activated Bpl I yields complete cleavage of DNA.
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PMID:BplI, a new BcgI-like restriction endonuclease, which recognizes a symmetric sequence. 935 50

Thermus species YS45 harbors two small cryptic plasmids of 5.8 (pTsp45s) and approximately 12 kb (pTsp45I). Plasmid pTsp45s has been entirely sequenced, revealing three significant ORFs. In addition to a previously reported thermophilic plasmid-encoded replication protein (Rep), pTsp45s contains two genes for the Tsp45I methyltransferase (M.Tsp45I) and restriction endonuclease (Tsp45I). These two converging genes (tsp45IM and tsp45IR) overlap by 4 bp at their stop codons within an XbaI site. M.Tsp45I (413 aa, 47.0 kDa, recognizing 5'-GTSAC-3') is highly homologous to other m6A-methyltransferases, especially M.EcaI (recognizing 5'-GGTNACC-3'). Tsp45I (332 aa, 37.4 kDa, cleaving 5'-/GTSAC-3') is not homologous to M.Tsp45I, or to other restriction endonucleases. Recombinant Tsp45I is stably produced in E. coli, and cleaves DNA at 65 degrees C with the same specificity as the native enzyme. Therefore, the thermophilic Tsp45I restriction-modification system is plasmid-borne within its native host.
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PMID:The Tsp45I restriction-modification system is plasmid-borne within its thermophilic host. 942 49


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