EcoRV

EcoRV Recombinant E. coli carrying EcoRV 5 -GAT4.ATC-3 ... 

A similar strategy was used to create plasmid pCP47. The insertion of a 10-His-tag was realized by cloning the tag at the end of the psbB gene via the restriction-sites BarnHI and EcoRV (see Fig. 2). In case of plasmid pCP47His, the selection-marker CmR was located downstream ofpsbB. 

Restriction endonucleases (EcoRV, EcoRI, Sncl, Kpnl, BamHl, and Xbal). Store at -20°C. 

Thousands of restriction endonucleases have been discovered in different bacterial species, and more than 100 different DNA sequences are recognized by one or more of these enzymes. The recognition sequences are usually 4 to 6 bp long and palindromic (see Fig. 8-20). Table 9-2 lists sequences recognized by a few type II restriction endonucleases. In some cases, the interaction between a restriction endonuclease and its target sequence has been elucidated in exquisite molecular detail for example, Figure 9-2 shows the complex of the type II restriction endonuclease EcoRV and its target sequence. 

Although they often share little sequence similarity and have quite different specificiities, many restriction enzymes have similar three-dimensional structures as well as mechanisms of action. This is true for the EcoRI, BamHl (Fig. 26-5),83/90 EcoRV,91/91a and C/r 101 enzymes,84 and presumably many others. The specifically shaped and tightly packed active sites in the enzyme-substrate complexes ensure specificity. For example, the EcoRV endonuclease cleaves DNA at its recognition site at least a million times faster than at any other DNA sequence.91 As mentioned in Chapter 12, restriction endonucleases require a metal ion, preferably Mg2+, and probably act via a hydroxyl ion generated from Mg2+-OH2 at the active site. Three conserved active site residues, Asp 91, Glu 111, and Lys 113, in the EcoRI endonuclease interact with the DNA near the cleavage site. Lys 113 is replaced by Glu 113 in the BamHl enzyme.83 90... 

Two enzymes (Kpnl and Pst in the list in Table 26-2) form 3 -cohesive ends rather than 5 -cohesive ends. In addition, there are three (Alul, EcoRV, and HaellT) that cut at the local twofold axis they form no cohesive ends but leave blunt ends (flush ends). Blunt end fragments are also much used in genetic engineering. "Linkers" that provide cohesive ends can be added.119 The Sfil endonuclease cuts between two 4-bp palindromes in a 13-bp recognition sequence (Table 26-2).120... 

PIG10 1x lacZ, 1x RBS 1x OmpA scFv no EcoRV -EcoRl myc full amber, supE strair 96... 

Reinhard B, Sheikholeslami S, Mastroianni A, Alivisatos AP, Liphardt J. Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes. Proc Natl Acad Sci USA 2007 104 2667-72. 

Fig. 13.2. Cleavage of the specific recognition sites by the type II restriction endonucleases EcoRV, EcoRI and Bg/I The cleavage reaction, which requires Mg2+ as cofactor, leads to 5 phosphate and 3 OH ends. While EcoRV cleavage results in blunt ends, EcoRI and Bg/I generate sticky ends with a 5 and 3 overhang, respectively.

Fig. 13.5. Structure of a specific EcoRV-DNA complex. The regions subjected to mutagenesis as described in Lanio et al (1998) are highlighted in red (amino acid residues 95 to 104), green (amino acid residues 180 to 184) and blue (amino acid residues 219 to 226). In B only the regions subjected to mutagenesis are displayed, together with the DNA.

Fig. 13.7. Cleavage experiments with prelinearized substrate and the EcoRV variant with the most pronounced preference for AT flanked recognition sites (Y95H K98E E99V N100T S183A Q224K). Substrate (4nM) (see Fig. 13.6) was incubated with enzyme (3 nM). Lane 0 = length marker, 1 - 13 = V, 3 , 5 , 6 , 10 , 30 , 60 , 90 , 120 , 150 , 170 - addition of new sub-...

Multiple efforts have been made to replace phosphorous-containing linkages with sulfur-containing isosteres in the context of enzyme inhibition. In a search for nonionic transition state analog inhibitors of restriction enzymes, Blattler et al. (34) found that nucleic acid duplexes that incorporate a dimethyl sulfone in place of a phosphodiester have distorted backbones similar to those in restriction enzyme bound DNA. Chimeric DNAs that incorporate sulfone hnkages were synthesized, and depending on the location of the dimethylene sulfone hnker, either between the first AT unit or the second AT unit in the EcoRV recognition site, values were 20 nM and 120... 

Figure 9.42. Greater Binding Energy of EcoRV Endonuclease Bound to Cognate Versus Noncognate Dna. The...

A. Jeltsch, J. Alves, G. Maass, and A. Pingoud. 1992. On the catalytic mechanism of EcoRI and EcoRV A detailed proposal based on biochemical results, structural data and molecular modelling FEES Lett 304 4-8. (PubMed)... 

F.K. Winkler, D.W. Banner, C. Oefiier, D. Tsemoglou, R.S. Brown, S.P. Heathman, R.K. Bryan, P.D. Martin, K. Petratos, and K.S. Wilson. 1993. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments EMBO J. 12 1781-1795. (PubMed)... 

D. Kostrewa and F.K. Winkler. 1995. Mg + binding to the active site of EcoRV endonuclease A crystallographic study of complexes with substrate and product DNA at 2 A resolution Biochemistry 34 683-696. (PubMed)... 

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