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)

Herpes simplex virus type 1 DNase (HSV-1 DNase) was expressed in insect cells by recombinant baculovirus (NPVUL12) and purified by a combination of anionic exchanger chromatography and gel filtration. Two polypeptides of 85 and 75 kD, whose ratio varied during purification, were induced 24 h after infection. The 75-kD protein was isolated and shown to possess catalytic activity. Gel filtration analysis indicated that the active form of the enzyme at an ionic strength of I = 0.3 is a dimeric protein with an apparent molecular weight of 130,000. The recombinant enzyme exhibited the overall characteristics of the native enzyme such as 5'-3' exonuclease and endonuclease activities with a preferred degradation of DNA. In the absence of extraneously added Mg2+, the enzyme was capable of removing mononucleotides from 5'-end-labeled DNA, but not from RNA and 3'-end-labeled DNA. The peculiar mechanism of double-strand DNA degradation suggests a specific role of HSV-1 DNase in DNA recombination processes during viral replication.
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PMID:Herpes simplex virus type 1 DNase: functional analysis of the enzyme expressed by recombinant baculovirus. 982 Aug 45

The DNA specificity subunit (HsdS) of type I restriction-modification enzymes is composed of two independent target recognition domains and several regions whose amino acid sequence is conserved within an enzyme family. The conserved regions participate in intersubunit interactions with two modification subunits (HsdM) and two restriction subunits (HsdR) to form the complete endonuclease. It has been proposed that the domains of the HsdS subunit have a circular organisation providing the required symmetry for their interaction with the other subunits and with the bipartite DNA target. To test this model, we circularly permuted the HsdS subunit of the type IB R-M enzyme EcoAI at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circularly permuted variants of HsdS that had termini located at equivalent positions in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity, suggesting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stable trimeric complex with HsdM, M2S1, which was fully active as a DNA methyltransferase. Gel electrophoretic mobility shift assays revealed that the HsdS protein alone was not able to form a specific complex with a 30-mer oligoduplex containing a single EcoAI recognition site. However, addition of stoichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular organisation of domains of the HsdS subunit. In addition, they suggest a possible role of HsdM subunits in the formation of this structure.
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PMID:The DNA recognition subunit of the type IB restriction-modification enzyme EcoAI tolerates circular permutions of its polypeptide chain. 983 17

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5, 6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage lambda DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.
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PMID:Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interaction. Type II restriction endonucleases as a model system. 1006 45

Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enterica serovar Enteritidis (S. enteritidis) is described. The procedure consists of plasmid DNA purification, its digestion with restriction endonuclease TaqI, electrophoresis, charge-coupled device camera scanning of the gels, and an analysis of the restriction patterns with the software Gel Manager. The system allowed us to analyse, in detail, results of plasmid profiling in more than 600 field strains of S. enteritidis. In addition to plasmid-free and virulence plasmid only containing strains, 15 additional plasmid types were detected. All the images and detailed protocols are available at the Web site http://www.clark.cz/vri/salmon.htm.
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PMID:Computer-assisted restriction endonuclease analysis of plasmid DNA in field strains of Salmonella enteritidis. 1034 65

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.
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PMID:Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis. 1052 May 96

BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRalpha gene, and the bslIRbeta gene. The alpha and beta subunits of BslI can be expressed independently in E. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (alphabeta), heterotetramers (alpha(2)beta(2)), and possibly oligomers in solution. Two beta subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (alpha(2)beta(2)). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is alpha(2)beta(2). M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the beta group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the alpha subunit of BslI endonuclease.
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PMID:Cloning, expression, and purification of a thermostable nonhomodimeric restriction enzyme, BslI. 1064 19

New restriction endonuclease (restrictase) Smil of type II was detected in the bacterial strain Streptococcus milleri. Cellular lysate enzyme cut T7 and adenovirus-2 DNAs at site 5'-ATTT decreases AAAT-3' but not lambda DNA which does not contain this sequence. Intense aeration inhibited the growth of S. milleri. The content of restrictase in the cells was the greatest during the logarithmic growth phase. A total of 20,000 units of Smil were isolated from 4 g of cells by cellular extract fractionation with ammonium sulfate and subsequent chromatography on columns with Bio Gel A 0.5 m, heparin agarose, and phosphocellulose. Purified enzyme cut the synthetic oligonucleotide duplex in the center of the recognized site 5'-ATTT decreases AAAT-3'. Smil restrictase is a true isoschisomer of rare-cutting Swal enzyme. Smil belongs to a small group of enzymes which recognize octanucleotide sites and can be used for large-block fragmentation of DNA. Comparison of specificities of rare-cutting and other restrictases suggests that the enzymes recognizing octanucleotides can evolutionally originate from enzymes recognizing both hexanucleotides and tetranucleotides.
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PMID:[New rare cutting restriction endonuclease SmII from Streptococcus milleri recognises 5'-ATTTAAAT-3']. 1070 87

We have performed mutational analyses of restriction endonuclease HindIII in order to identify the amino acid residues responsible for enzyme activity. Four of the seven HindIII mutants, which had His-tag sequences at the N-termini, were expressed in Escherichia coli, and purified to homogeneity. The His-tag sequence did not affect enzyme activity, whereas it hindered binding of the DNA probe in gel retardation assays. A mutant E86K in which Lys was substituted for Glu at residue 86 exhibited high endonuclease activity. Gel retardation assays showed high affinity of this mutant to the DNA probe. Surprisingly, in the presence of a transition metal, Mo(2+) or Mn(2+), the E86K mutant cleaved substrate DNA at a site other than HindIII. Substitution of Glu for Val at residue 106 (V106E), and Asn for Lys at residue 125 (K125N) resulted in a decrease in both endonucleolytic and DNA binding activities of the enzyme. Furthermore, substitution of Leu for Asp at residue 108 (D108L) abolished both HindIII endonuclease and DNA binding activities. CD spectra of the wild type and the two mutants, E86K and D108L, were similar to each other, suggesting that there was little change in conformation as a result of the mutations. These results account for the notion that Asp108 could be directly involved in HindIII catalytic function, and that the substitution at residue 86 may bring about new interactions between DNA and cations.
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PMID:Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity. 1081 Jan 60

Triple helix-forming oligonucleotides conjugated to a psoralen (psoTFO) have been designed to bind to three distinct purine-rich sequences within the human interstitial collagenase (MMP1) gene. Gel mobility shift assays indicate that these psoTFO bind to and photoreact with model target DNA sequences following ultraviolet A (UVA) irradiation. The dissociation constants for binding of the psoTFO to their targets range from 0.3 to 4 microM. Psoralen monoadducts with the purine-rich target strand and interstrand crosslinks are efficiently formed on targets containing either 5'-ApT-3' or 5'-TpA-3' sequences adjacent to the TFO binding sequence. The dependence of adduct formation on UVA dose has provided quantitative estimates of the overall rate constants for psoralen monoadduct and crosslink formation in the presence of a TFO. When psoralen is tethered to a TFO, the rate of monoadduct formation exceeds that of crosslinking for all sequences studied. This contrasts with the relatively low rate of monoadduct formation that has been reported for free psoralens, suggesting that the bound TFO facilitates the initial photochemistry that generates monoadducts, but does not significantly affect interstrand crosslink formation. psoTFO and UVA treatment inhibit DNA cleavage by a restriction endonuclease when the psoralen covalently reacts directly at the endonuclease site. The particular TFO studied do not completely inhibit endonuclease activity when they are noncovalently bound or when the covalent psoralen adduct does not coincide with the endonuclease site. Our findings confirm that TFO are capable of directing psoralen photoadducts to specific DNA targets and suggest that TFO can significantly modulate psoralen photoreactivity and DNA-protein interactions.
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PMID:Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides. 1098 98

N.BstNBI is a nicking endonuclease that recognizes the sequence GAGTC and nicks the top strand preferentially. The Type IIs restriction endonucleases PleI and MlyI also recognize GAGTC, but cleave both DNA strands. Cloning and sequencing the genes encoding each of these three endonucleases discloses significant sequence similarities. Mutagenesis studies reveal a conserved set of catalytic residues among the three endonucleases, suggesting that they are closely related to each other. Furthermore, PleI and MlyI contain a single active site for DNA cleavage. The results from cleavage assays show that the reactions catalyzed by PleI and MlyI are sequential two step processes. The double-stranded DNA is first nicked on one DNA strand and then further cleaved on the second strand to form linear DNA. Gel filtration analysis shows that MlyI dimerizes in the presence of a cognate DNA and Ca(2+) whereas N.BstNBI remains a monomer, implicating dimerization as a requisite for the second strand cleavage. We suggest that N.BstNBI, MlyI and PleI diverged from a common ancestor and propose that N.BstNBI differs from MlyI and PleI in having an extremely limited second strand cleavage activity, resulting in a site-specific nicking endonuclease.
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PMID:The nicking endonuclease N.BstNBI is closely related to type IIs restriction endonucleases MlyI and PleI. 1141 Jun 56


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