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1.
To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented. 相似文献
2.
Primary sequence of the EcoRII endonuclease and properties of its fusions with beta-galactosidase 总被引:14,自引:0,他引:14
The EcoRII endonuclease cleaves DNA containing the sequence CC(A/T)GG before the first cytosine. The methylation of the second cytosine in the sequence by either the EcoRII methylase or Dcm, a chromosomally coded protein in Escherichia coli, inhibits the cleavage. The gene for the EcoRII endonuclease was mapped by analysis of derivatives containing linker insertions, transposon insertions, and restriction fragment deletions. Surprisingly, plasmids carrying the wild-type endonuclease gene and the EcoRII methylase gene interrupted by transposon insertions appeared to be lethal to dcm+ strains of E. coli. We conclude that not all the EcoRII/Dcm recognition sites in the cellular DNA are methylated in dcm+ strains. The DNA sequence of a 1650-base pair fragment containing the endonuclease gene was determined. It revealed an open reading frame that could code for a 45.6-kDa protein. This predicted size is consistent with the known size of the endonuclease monomer (44 kDa). The endonuclease and methylase genes appear to be transcribed convergently from separate promoters. The reading frame of the endonuclease gene was confirmed at three points by generating random protein fusions between the endonuclease and beta-galactosidase, followed by an analysis of the sequence at the junctions. One of these fusions is missing 18 COOH-terminal amino acids of the endonuclease but still displays significant ability to restrict incoming phage in addition to beta-galactosidase activity. No striking similarity between the sequence of the endonuclease and any other protein in the PIR data base was found. The knowledge of the primary sequence of the endonuclease and the availability of the various constructs involving its gene should be helpful in the study of the interaction of the enzyme with its substrate DNA. 相似文献
3.
Nucleotide sequence of the EcoRII restriction endonuclease gene 总被引:3,自引:0,他引:3
The nucleotide sequence of a 1394 basepair (bp) DNA fragment containing the EcoRII restriction endonuclease (R.EcoRII) gene was determined. The endonuclease gene is 1206 bp in length (predicted 402 amino acids (aa) and Mr = 45 178) and is separated by 33 bp from the EcoRII modification methylase (M.EcoRII) gene. The EcoRII restriction-modification system has a tail-to-tail organization of the two genes. 相似文献
4.
Mücke M Lurz R Mackeldanz P Behlke J Krüger DH Reuter M 《The Journal of biological chemistry》2000,275(39):30631-30637
EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding. 相似文献
5.
M N Vinogradova E S Gromova A A Purmal' V G Kosykh Z A Shabarova 《Molekuliarnaia biologiia》1986,20(5):1329-1336
As shown by a nitrocellulose filter binding assay, in the absence of Mg2+ EcoRII restriction endonuclease binds specifically to a set of synthetic concatemer DNA duplexes of varying chain length, containing natural and modified recognition sites of this enzyme. The binding of the substrates with the central AT, TT or AA-pair in the recognition site decreases at AT greater than TT much greater than AA. Substitution of the pyrophosphate bond at the cleavage site for the phosphodiester or phosphoramide bond produces little influence on the stability of the complexes. The affinity of the enzyme for nonspecific sites is two orders of magnitude less than that for the specific EcoRII sequences. Equilibrium association constant for a substrate with one recognition site is 3.9 X 10(8) M-1. Addition of Mg2+ leads to the destabilization of the EcoRII endonuclease complex with DNA duplex, containing pyrophosphate bonds. The dissociation rate constants and the lifetime of the EcoRII endonuclease--synthetic substrates complexes have been determined. 相似文献
6.
V G Kosykh Iu A Mantsygin N V Sviatukhina I V Vitenene Ia I Bur'ianov 《Biokhimii?a (Moscow, Russia)》1988,53(9):1474-1478
Monoclonal antibodies against EcoRII endonuclease were obtained after immunization of two BALB/c mice with a homogeneous enzyme prepared by conventional methods. IgG from ascitic fluid was purified and coupled to CNBr-activated Sepharose 4B to give a specific column used to isolate EcoRII endonuclease. The isolated EcoRII endonuclease produced a single band during SDS gel electrophoresis. 相似文献
7.
The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5'CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two-domain structure that enables this particular mode of protein-DNA interaction. The C-terminal domain is a new restriction endonuclease, EcoRII-C. In contrast to the wild-type enzyme, EcoRII-C cleaves DNA specifically at single 5'CCWGG sites. Moreover, substrates containing two or more cooperative 5'CCWGG sites are cleaved much more efficiently by EcoRII-C than by EcoRII. The N-terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5'CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA-binding domain to enable the interaction with two 5'CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons. 相似文献
8.
Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease. Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed. When expressed in E. coli, all these plasmids displayed EcoRII endonuclease activity. We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein. This mutant protein had no EcoRII endonuclease activity. The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site. However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease. 相似文献
9.
Michal Szczepek Petra Mackeldanz Elisabeth Möncke-Buchner Jürgen Alves Detlev H. Krüger Monika Reuter 《Molecular microbiology》2009,72(4):1011-1021
Bacterial restriction endonuclease EcoRII requires two recognition sites to cleave DNA. Proteolysis of EcoRII revealed the existence of two stable domains, EcoRII-N and EcoRII-C. Reduction of the enzyme to its C-terminal domain, EcoRII-C, unleashed the enzyme activity; this truncated form no longer needed two recognition sites and cleaved DNA much more efficiently than EcoRII wild-type. The crystal structure of EcoRII showed that probably the N-terminal domain sterically occludes the catalytic site, thus apparently controlling the cleavage activity. Based on these data, EcoRII was the first restriction endonuclease for which an autoinhibition mechanism as regulatory strategy was proposed. In this study, we probed this assumption and searched for the inhibitory element that mediates autoinhibition. Here we show that repression of EcoRII-C is achieved by addition of the inhibitory domain EcoRII-N or by single soluble peptides thereof in trans . Moreover, we perturbed contacts between the N- and the C-terminal domain of EcoRII by site-directed mutagenesis and proved that β-strand B1 and α-helix H2 are essential for autoinhibition; deletion of either secondary structural element completely relieved EcoRII autoinhibition. This potent regulation principle that keeps EcoRII enzyme activity controlled might protect bacteria against suicidal restriction of rare unmodified recognition sites in the cellular genome. 相似文献
10.
E A Kubareva E S Gromova T S Oretskaia Z A Shabarova 《Bioorganicheskaia khimiia》1987,13(9):1205-1211
Interaction of the EcoRII restriction endonuclease with a set of 30-membered substrates having structural anomalies in the recognition site (decreases CCT/AGG) and in adjacent sequences has been studied. A nick in the centre of the EcoRII recognition site between dC and dA residues slows down hydrolysis of the nonmodified strand, whereas the modified one is not cleaved. Removal of the phosphate group from the nick in this substrate does not alter the rate of the cleavage. The absence of one of the phosphate groups in the flanking sequence at a two-base-pair "distance" from the recognition site slows down the enzymatic hydrolysis. Removal of dA or dT out of the EcoRII recognition site blocks the enzymatic reaction. It appears that EcoRII does not interact with the phosphate group between dC and dA residues in the recognition site. Suggestions are made concerning possible contacts of the EcoRII restriction endonuclease with dA- and dT-residues of the recognition site and with the sugar-phosphate backbone of the adjacent nucleotide sequences. 相似文献
11.
Waleron K Waleron M Osipiuk J Podhajska AJ Lojkowska E 《Journal of applied microbiology》2006,100(2):343-351
AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples. 相似文献
12.
The X-ray structure for the type IIE EcoRII restriction endonuclease has been resolved [X.E. Zhou, Y. Wang, M. Reuter, M. Mucke, D.H. Kruger, E.J. Meehan and L. Chen. Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold. J. Mol. Biol. 335 (2004) 307-319.], but the structure of the R.EcoRII-DNA complex is still unknown. The aim of this article was to examine the structure of the pre-reactive R.EcoRII-DNA complex in solution by fluorescence spectroscopy. The structure for the R.EcoRII-DNA complex was resolved by determining the fluorescence resonance energy transfer (FRET) between two fluorescent dyes, covalently attached near the EcoRII recognition sites, that were located at opposite ends of a lengthy two-site DNA molecule. Analysis of the FRET data from the two-site DNA revealed a likely model for the arrangement of the two EcoRII recognition sites relative to each other in the R.EcoRII-DNA complex in the presence of Ca(2+) ions. According to this model, the R.EcoRII binds the two-site DNA and forms a DNA loop in which the EcoRII recognition sites are 20+/-10 A distant to each other and situated at an angle of 70+/-10 degrees. 相似文献
13.
14.
Mücke M Pingoud V Grelle G Kraft R Krüger DH Reuter M 《The Journal of biological chemistry》2002,277(16):14288-14293
The EcoRII homodimer engages two of its recognition sequences (5'-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodo-pyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5'-C of the 5'-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5'-C of the 5'-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Tryptic digestion of free and of cross-linked EcoRII, followed by high performance liquid chromatography (HPLC) separation of the individual peptides and Edman degradation, identified amino acids 25-49 of EcoRII as the cross-linking peptide. Mutational analysis of the electron-rich amino acids His(36) and Tyr(41) of this peptide indicates that Tyr(41) is the amino acid involved in the cross-link and that it therefore contributes to specific DNA recognition by EcoRII. 相似文献
15.
Pingoud V Kubareva E Stengel G Friedhoff P Bujnicki JM Urbanke C Sudina A Pingoud A 《The Journal of biological chemistry》2002,277(16):14306-14314
The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype. 相似文献
16.
Oligonucleotide duplexes containing CC(A/T)GG stimulate cleavage of refractory DNA by restriction endonuclease EcoRII 总被引:3,自引:0,他引:3
Some DNA species are resistant towards the restriction endonuclease EcoRII despite the presence of unmodified recognition sites. We show that 14 base-pair oligonucleotide duplexes containing the EcoRII recognition site 5'-CC(A/T)GG are cleaved by this enzyme and are able to stimulate EcoRII cleavage of such resistant DNA molecules (e.g. DNA of bacterial virus T3). A direct correlation between the concentration of oligonucleotide duplex molecules and the degree of EcoRII digestion of the primarily resistant DNA is observed. This indicates a stoichiometric rather than a catalytic mode of enzyme activation. An excess of DNA devoid of EcoRII sites ('non-site' DNA, e.g. MvaI-digested T7 DNA) does not interfere with the activity of EcoRII. 相似文献
17.
Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages. 总被引:3,自引:3,他引:0
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A A Yolov E S Gromova E A Kubareva V K Potapov Z A Shabarova 《Nucleic acids research》1985,13(24):8969-8981
Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate. 相似文献
18.
O V Petrauskene E A Kubareva E S Gromova Z A Shabarova 《European journal of biochemistry》1992,208(3):617-622
The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11-14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiencies or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate. 相似文献
19.
E S Gromova A A Elov E A Kubareva V G Metelev Z A Shabarova 《Molekuliarnaia biologiia》1986,20(1):29-40
A set of DNA duplexes with repeated EcoRII, EcoRI and AluI restriction endonuclease recognition sites in which EcoRII scissile phosphodiester bonds were replaced by phosphoramide or uncleavable pyrophosphate bonds have been synthesized. Endonuclease EcoRII was found not to cleave the substrate at the phosphoramide bond. The substrates containing non-nydrolysable pyrophosphate or phosphoramide bonds in one of the chains of EcoRII recognition sites were used to show that this enzyme is able to catalyze single-strand scissions. These scissions occur both in dA- and dT-containing chains of the recognition site. Endonuclease EcoRII interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of the other. Synthesized DNA-duplexes are cleaved specifically by EcoRI and AluI endonucleases, this cleavage being retarded if the modified bonds are in the recognition site (EcoRI) or flank it (AluI). For EcoRII and AluI this effect is more pronounced in the case of substrates with pyrophosphate bonds than with the phosphoramide ones. 相似文献
20.
M V Vinogradova E S Gromova V G Kosykh Ia I Bur'ianov Z A Shabarova 《Molekuliarnaia biologiia》1990,24(3):847-850
EcoRII restriction endonuclease binding site has been determined on the basis of comparison of the binding parameters of the enzyme with synthetic DNA-duplexes of concatemer type containing a different number of EcoRII recognition sites. It has been shown that it consists of 21 +/- 1 base pairs. 相似文献