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)

In the SalI system, endonuclease activity can be only achieved in the presence of a functional modification gene. Thus, the DNA methyltransferase is involved in the control of restriction. By fusion of the restriction gene of the SalI system to the modification gene of the isospecific HgiDII system a hybrid type II restriction-modification system was created. Although in the hybrid situation the level of endonuclease activity was significantly lower than in the natural system, the HgiDII modification enzyme clearly supports SalI restriction. The mechanism by which the two isospecific methyltransferases control restriction is currently under study.
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PMID:Establishment of a hybrid SalI-HgiDII type II restriction-modification system. 962 60

Ecl18kI is a type II restriction-modification system isolated from Enterobacter cloaceae 18kI strain. Genes encoding Ecl18kI methyltransferase (M.Ecl18kI) and Ecl18kI restriction endonuclease (R.Ecl18kI) have been cloned and expressed in Escherichia coli. These enzymes recognize the 5'.../CCNGG...3' sequence in DNA; M.Ecl18kI methylates the C5 carbon atom of the inner dC residue and R.Ecl18kI cuts DNA as shown by the arrow. The restriction endonuclease and the methyltransferase were purified from E. coli B834 [p18Ap1] cells to near homogeneity. The restriction endonuclease is present in the solution as a tetramer, while the methyltransferase is a monomer. The interactions of M.Ecl18kI and R.Ecl18kI with 1,2-dideoxy-D-ribofuranose containing DNA duplexes were investigated. The target base flipping-out mechanism is applicable in the case of M.Ecl18kI. Correct cleavage of the abasic substrates by R.Ecl18kI is accompanied by non-canonical hydrolysis of the modified strand.
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PMID:The Ecl18kI restriction-modification system: cloning, expression, properties of the purified enzymes. 974 1

The class-IIS restriction endonuclease, R.MmeI, was isolated from Methylophilus methylotrophus. It was originally described as a monomeric enzyme, with the native Mr 105000+/-7000, which did not cleave DNA efficiently [Boyd et al. (1986) Nucleic Acids Res. 14, 5255-5274; Tucholski et al. (1995) Gene 157, 87-92]. However, it was discovered that R.MmeI endonucleolytic activity is enhanced by S-adenosyl-l-methionine (AdoMet) and sinefungin, an analogue of AdoMet. Surprisingly, the purified R.MmeI endonuclease was found to have a second enzymatic activity, namely methylation of the adenine residue to N6-methyladenine in the top strand of the MmeI-recognition sequence, 5'-TCCR*AC-3' (*A=meA. The R.MmeI methylating activity requires AdoMet and is increased in the presence of several divalent cations, 20-fold by Mg2+ or Ca2+, and less by Mn2+, Zn2+ and Co2+; however, methylation is inhibited entirely by sinefungin, at concentrations above 9microM. The latter observation shows that the enhancing effect of AdoMet or sinefungin on the DNA cleavage was not related to the process of DNA methylation. Furthermore, a second component of the MmeI restriction-modification system, a M.MmeI methyltransferase, was isolated and purified. The M.MmeI protein was found to have an Mr of 48000+/-2000 (under denaturing conditions) and to methylate both adenine residues (*A) in the MmeI-recognition sequence 5'-TCCR*AC-3'/3'-*AGGYTG-5'. Methylation of the top strand does not inhibit the DNA cleavage by R.MmeI, whereas methylation of both DNA strands blocks the cleavage process.
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PMID:Two intertwined methylation activities of the MmeI restriction-modification class-IIS system from Methylophilus methylotrophus. 985 52

The genes encoding the ApaLI (5'-GTGCAC-3'), NspI (5'-RCATGY-3'), NspHI (5'-RCATGY-3'), SacI (5'-GAGCTC-3'), SapI (5'-GCTCTTCN1-3', 5'-N4GAAGAGC-3') and ScaI (5'-AGTACT-3') restriction-modification systems have been cloned in E. coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.
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PMID:Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli. 986 76

In contrast to many type II restriction enzymes, dimeric proteins that cleave DNA at individual recognition sites 4-6 bp long, the SfiI endonuclease is a tetrameric protein that binds to two copies of an elongated sequence before cutting the DNA at both sites. The mode of action of the SfiI endonuclease thus seems more appropriate for DNA rearrangements than for restriction. To elucidate its biological function, strains of Escherichia coli expressing the SfiI restriction-modification system were transformed with plasmids carrying SfiI sites. The SfiI system often failed to restrict the survival of a plasmid with one SfiI site, but plasmids with two or more sites were restricted efficiently. Plasmids containing methylated SfI sites were not restricted. No rearrangements of the plasmids carrying SfiI sites were detected among the transformants. Hence, provided the target DNA contains at least two recognition sites, SfiI displays all of the hallmarks of a restriction-modification system as opposed to a recombination system in E. coli cells. The properties of the system in vivo match those of the enzyme in vitro. For both restriction in vivo and DNA cleavage in vitro, SfiI operates best with two recognition sites on the same DNA.
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PMID:DNA restriction dependent on two recognition sites: activities of the SfiI restriction-modification system in Escherichia coli. 1009 90

Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.
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PMID:On the structure and operation of type I DNA restriction enzymes. 1039 Mar 54

Structural studies of the proteins of the BstVI restriction-modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 degrees C and 55 or 60 degrees C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3-5 microM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 microM at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 degrees C and 55 degrees C. These results were interpreted in terms of differences in the structural characteristics of these restriction-modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 degrees C and at the temperature at which they are most active.
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PMID:Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy. 1042 88

Eco KI, a type I restriction enzyme, specifies DNA methyltransferase, ATPase, endonuclease and DNA translocation activities. One subunit (HsdR) of the oligomeric enzyme contributes to those activities essential for restriction. These activities involve ATP-dependent DNA translocation and DNA cleavage. Mutations that change amino acids within recognisable motifs in HsdR impair restriction. We have used an in vivo assay to monitor the effect of these mutations on DNA translocation. The assay follows the Eco KI-dependent entry of phage T7 DNA from the phage particle into the host cell. Earlier experiments have shown that mutations within the seven motifs characteristic of the DEAD-box family of proteins that comprise known or putative helicases severely impair the ATPase activity of purified enzymes. We find that the mutations abolish DNA translocation in vivo. This provides evidence that these motifs are relevant to the coupling of ATP hydrolysis to DNA translocation. Mutations that identify an endonuclease motif similar to that found at the active site of type II restriction enzymes and other nucleases have been shown to abolish DNA nicking activity. When conservative changes are made at these residues, the enzymes lack nuclease activity but retain the ability to hydrolyse ATP and to translocate DNA at wild-type levels. It has been speculated that nicking may be necessary to resolve the topological problems associated with DNA translocation by type I restriction and modification systems. Our experiments show that loss of the nicking activity associated with the endonuclease motif of Eco KI has no effect on ATPase activity in vitro or DNA translocation of the T7 genome in vivo.
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PMID:The DNA translocation and ATPase activities of restriction-deficient mutants of Eco KI. 1052 5

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.
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PMID:Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer. 1061 66

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C. PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C. PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5' ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C. PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C. PvuII binding, overlapping the -10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.
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PMID:Role and mechanism of action of C. PvuII, a regulatory protein conserved among restriction-modification systems. 1062 96


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