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 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 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

The physical map of the plasmid pSACII1 carrying the genes of restriction-modification system Eco29kI (isoschizomer of SacII) was determined. The cloning of the Eco29kI endonuclease and methylase genes into the plasmid vector pUC129 produced recombinant strain Escherichia coli K802[pECO29A15] with Eco29kI synthesis level about 100 times higher than in the parent strain. The restriction endonuclease was purified from Escherichia coli K802 [pECO29A15] cells to near homogeneity using column chromatography sequentially on phosphocellulose, hydroxyapatite, and heparin-Sepharose and rechromatography on phosphocellulose. Biochemical characterization of the homogeneous R Eco29kI is given. The enzyme has molecular mass 24.5 kD and is present in the solution as a monomer.
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PMID:Isolation of a strain overproducing endonuclease Eco29kI: purification and characterization of the homogeneous enzyme. 933 65

A neutral Mg(2+)-dependent deoxyribonuclease from the Colorado potato beetle was isolated and characterized in physicochemical terms. An electrophoretically homogeneous preparation of the enzyme was obtained using salt fractionation, Sephadex G-100 gel filtration, and subsequent preparative isoelectrofocusing in an Ultrodex layer. The molecular weight of the purified DNase preparation (with a purification degree of 104) and its isoelectric point were 100 kD and 9.1, respectively. The enzyme activity was maximal at pH 7.2 and 46 degrees C in the presence of 10 mM Mg2+. The DNase of the Colorado beetle preferentially hydrolysed denatured DNA via the endonuclease pathway, degrading the substrate to oligonucleoside-3'-phosphates. As far as the physical and chemical properties are concerned, this Colorado beetle DNase seems different from previously investigated DNases of other insect species.
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PMID:Isolation, purification, and characterization of a neutral Mg(2+)-dependent deoxyribonuclease of the Colorado potato beetle Leptinotarsa decemlineata Say. 936 Mar 4

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

The Eco29kI restriction-modification system (RMS2) has been found to be localized on the plasmid pECO29 occurring naturally in the Escherichia coli strain 29k (Pertzev, A.V., Ruban, N.M., Zakharova, M.V., Beletskaya, I.V., Petrov, S.I., Kravetz, A.N., Solonin, A.S., 1992. Eco29kI, a novel plasmid encoded restriction endonuclease from Escherichia coli. Nucleic Acids Res. 20, 1991). The genes coding for this RMS2, a SacII isoschizomer recognizing the sequence CCGCGG have been cloned in Escherichia coli K802 and sequenced. The DNA sequence predicts the restriction endonuclease (ENase) of 214 amino acids (aa) (24,556 Da) and the DNA-methyltransferase (MTase) of 382 aa (43,007 Da) where the genes are separated by 2 bp and arranged in tandem with eco29kIR preceding eco29kIM. The recombinant plasmid with eco29kIR produces a protein of expected size. MEco29kI contains all the conserved aa sequence motifs characteristic of m5C-MTases. Remarkably, its variable region exhibits a significant similarity to the part of the specific target-recognition domain (TRD) from MBssHII--multispecific m5C-MTase (Schumann, J.J., Walter, J., Willert, J., Wild, C., Koch D., Trautner, T.A., 1996. MBssHII: a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties. J. Mol. Biol. 257, 949-959), which recognizes five different sites on DNA (HaeII, MluI, Cfr10I, SacII and BssHII), and the comparison of the nt sequences of its variable regions allowed us to determine the putative TRD of MEco29kI.
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PMID:Cloning and sequence analysis of the plasmid-borne genes encoding the Eco29kI restriction and modification enzymes. 952 60

Although the product of the UL12 gene of herpes simplex virus type 1 (HSV-1) has been shown to possess both exonuclease and endonuclease activities in vitro, and deletion of most of the gene within the viral genome results in inefficient production and maturation of infectious virions, the function of the deoxyribonuclease (DNase) activity per se in virus replication remains unclear. In order to correlate the in vitro and in vivo activities of the protein encoded by UL12, mutant proteins were tested for nuclease activity in vitro by a novel hypersensitivity cleavage assay and for their ability to complement the replication of a DNase null mutant, AN-1. Rabbit reticulocyte lysates programmed with wild-type UL12 RNA cleaved at the same sites cleaved by purified HSV-1 DNase, but distinct from those cleaved by DNase 1 or micrococcal nuclease. All mutants which lacked DNase activity in vitro also failed to complement the replication of AN-1 in nonpermissive cells. Likewise, all mutants which contained HSV-1 DNase activity, as detected by the hypersensitivity cleavage assay, were capable of complementing the replication of the DNase null mutant, though to varying extents. Of particular note was the d1-126 mutant protein, which, despite having the same specific activity as the wild-type enzyme in vitro, complemented the replication of AN-1 significantly less than the wild-type protein. The results suggest that DNase activity per se is required for efficient replication of HSV-1 in vivo. However, residues, including the N-terminal 126 amino acids, which are dispensable for enzymatic activity in vitro may facilitate the accessibility or activity of the protein in vivo.
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PMID:Structure-function analysis of the herpes simplex virus type 1 UL12 gene: correlation of deoxyribonuclease activity in vitro with replication function. 952 34

A deoxyribonuclease that is secreted from an insectivorous plant Drosera adelae was partially purified by column chromatography. The enzyme acted as an endonuclease on double-stranded DNA and generated oligonucleotides with 3' hydroxyl and 5' phosphate ends. The activity of the enzyme was high in the range from pH 3.5 to 5.0. The enzyme seemed to require divalent cations for maximum activity.
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PMID:Deoxyribonuclease secreted from an insectivorous plant Drosera adelae. 958 32

The type I restriction-modification system EcoR124I recognizes and binds to the split DNA recognition sequence 5'-GAAN(6)RTCG-3'. The methyltransferase, consisting of HsdM and HsdS subunits with the composition M2S, can interact with one or more subunits of the HsdR subunit to form the endonuclease. The interaction of the methyltransferase with HsdR has been investigated by surface plasmon resonance, showing that there are two non-equivalent binding sites for HsdR which differ in binding affinity by at least two orders of magnitude. DNA footprinting experiments using Exonuclease III suggest that the addition of HsdR to the methyltransferase (at a stoichiometry of either 1:1 or 2:1) increases the stability of the resulting DNA-protein complex but does not increase the size of the footprint. More extensive in situ footprinting experiments using copper-phenanthroline on the DNA-protein complexes formed by M2S, R1M2S and R2M2S also show no difference in the detailed cleavage pattern, with approximately 18 nucleotides protected on both strands in each complex. Thus the HsdR subunit(s) of the endonuclease stabilise the interaction of the M2S complex with DNA, but do not directly contribute to DNA binding. In addition, the thymidine nucleotide in the tetranucleotide recognition sequence GTCG is hyper-reactive to cleavage in each case, suggesting that the DNA structure in this region is altered in these complexes.
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PMID:Protein-protein and protein-DNA interactions in the type I restriction endonuclease R.EcoR124I. 962 43

Sequence analysis of the BcnI restriction-modification system revealed the presence of an open reading frame encoding a second cytosine-N4 methyltransferase, M.BcnIA, in the vicinity of the genes specifying the previously characterized cytosine-N4 methyltransferase M.BcnIB and restriction endonuclease R.BcnI. Both methyltransferases were purified from the E. coli cells expressing the individual genes, and their enzymatic efficiencies in vitro were compared with a variety of DNA substrates. Both enzymes act on 5'-CC(C/G)GG-3' sites in double-stranded DNA, however, M.BcnIA can also, with a comparable efficiency, modify the specific targets in single-stranded DNA. The biological significance of the presence of the tandem methyltransferases in the BcnI system is discussed.
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PMID:A pair of single-strand and double-strand DNA cytosine-N4 methyltransferases from Bacillus centrosporus. 962 56


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