Restriction EcoRV

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... 

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.

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... 

Some enzymes discriminate between potential substrates by binding them Wxth different affinities. Others may bind many potential substrates but promote chemical reactions efficiently only on specific molecules. Restriction endonucleases such as EcoRV endonuclease employ... 

The most often used restriction endonucleases for staphylococci are HinAlll, Clal (Tenover et al. 1994), EcoRI (Blumberg et al. 1992), EcoRV or Kpnl (Blanc et al. 1994). The fragments containing specific sequences are then detected using a labelled piece of homologous DNA as a probe. The most often used is DNA complementary to 16S and 23S rRNA isolated from Escherichia coli and commercially available for ribotyping (Falkinham 1994 Pfaller and Hollis 2004). 

The role of the divalent metal ions present in natural phosphodiesterases became clear in bovine pancreatic deoxyribonuclease I (DNase I), the first endonuclease structure determined by X-ray crystallography. The nucleophilic attack of a water molecule activated by a histidine residue is facilitated by the interaction of a calcium ion with the phosphate group to be cleaved (291). Glutamic and aspartic residues involved in magnesium binding have been identified in the crystal structure of four type II restriction enzymes EcoRl (292), EcoRV (293), Pvull (294), and BamHl (295), as well as in that of the repair... 

Figure 4.31 The importance of linear diffusion on the kinetics of cleavage by EcoRV. EcoRV recognizes and cleaves GAT ATC sites on DNA. The overall rate of cleavage was determined for PCR-amplified DNA sequences of different length, but each containing one restriction site, (a) Model for association and one-dimensional diffusion, (b) Experimental data for relative rate of cleavage for long DNA vs short DNA (26 bp) [133].

Jeltsch, A., et al.. Linear diffusion of the restriction endonuclease EcoRV on DNA is essenticd for the in vivo function of the enzyme. EMBO Journal, 1996, 15(18), 5104-5111. 

Jeltsch, A. and A. Pingoud, Kinetic characterization of linear diffusion of restriction endonuclease EcoRV on DNA. Biochemistry, 1998, 37, 2160-2169. 

Figure 10. Map of the yeast ILV2 gene within a cloned DNA segment. Letters indicate restriction enzyme cleavage sites C, Clal V, EcoRV B, Bglll K, Kpnl R, EcoRI P, PvuII H, Hindlll. Deletions are indicated by bars, whereas lollipops indicate the sites of Tn 5 insertions. Mutations represented by filled symbols destroy ILV2 activity, whereas those depicted as open symbols do not affect ILV2 function. The extent of ILV2 mRNA is indicated by the wavy arrow.

Figure 2. Restriction maps of the regions cloned from P. syringae and P fluorescens. Solid bars indicate the region deleted for the marker exchange experiments. A Ball B BamHI C Bglll E EcoRI F EcoRV H Hpal K Kpnl L SphI P ...

On average, hove many EcoRV restriction sites would you expect in the genome of E. coli The genome is 4.6 x 10 base pairs and its composition is approximately 50% G + C. 

Partial restriction map of the EcoRV/EcoRI fragment containing psbDI and part of osbC cloned in M13mp19. The total length of the construct is 10 kbp. The psbD and psbC sequences from Svnechocvstis 6803 have been published [20,21]. 

In the following sections, some of the approaches and applications described in this article are illustrated using three case studies from our own work. We begin with a mechanistic study of the bacterial organomercurial lyase, MerB, using a quantum chemical cluster method, followed by a QM/MM free energy simulation of the dual-specificity phosphatase, Cdc25B, and a dual QM and QM/MM study of the restriction enzyme EcoRV. 

Case Study 3 The Restriction Enzyme, EcoRV. The following is a synopsis of Imhof and co-workers (2009) Catalytic Mechanism of DNA Backbone Cleavage by the Restriction Enzyme EcoRV (173). Here, the emphasis is on the exploration of many different pathways at a relatively low level of theory... 

Fig. 4. Energetically most favorable pathways for the phosphate hydrolysis reaction catalyzed by the restriction enzyme EcoRV. Energies (in kcal/mol) of intermediate states are given in blue and transition state energies are shown in red. The three different pathways are distinguished by colored arrows, and the corresponding atom movements are indicated with arrows of the respective color. The most likely pathway is the dissociative route R-aO-dl-d2Asp-d202-P, labeled in light blue and shown in the center. Reprinted with permission from Biochemistry, 48, 9061-9075 (2009). Cop3rright 2009 American Chemical Society.

Figure 1. Southern blot analysis of soybean genomic DNA. Probes used are. aCT cDNA (A) and BCCP cDNA (B). Restriction enzymes used for digestions are (1) -Nde1 (2) - Asel (3) - Bsu36 1 (4) EcoRV (5) - Avr II (6) - Afl II. All hybridizations were run at 60°C. Positions of lambda/Hindlll markers are shown. Presented data suggest multiple gene patterns for both subunits.

---