ECoRI restriction endonuclease

Blasiak J, Kowalik J. 1998. Interaction between organophosphoms compounds and DNA assayed by the restriction endonuclease EcoRI. Acta Univ Lodz Folia Biochim Biophys 13 31-67. 

This Is the DNA sequence recognized by the restriction endonuclease EcoRI. Rgure 1-6-1. EcoRI Recognition Sequence... 

Recognition sequence of restriction endonuclease EcoRI shows two-fold rotational symmetry. 

Restriction endonuclease EcoRI is able to cut a chain in dsDNA which has a chiral phophorothioate group at the specific cleavage site.838 Tire reaction occurs with inversion of configuration at phosphorus, suggesting direct in-line attack by a hydroxyl ion generated from H20. 

DNA Sequence Analysis. DNA from AgtlO clone 14-1 was digested with restriction endonuclease EcoRI and cloned into M13 mpll (18). The bacteriophage M13 derivatives were constructed using methods described by Messing (19). Both orientations of the inserts were represented in independent clones that were sequenced by the dideoxy method (20). 

Fig. 6.18. Actions of restriction endonucleases EcoRI, Psfl, and Sma at their recognition sequences. Note that EcoRI and Pst enzymes produce "sticky ends, with overlapping sequences, whereas Smal produces blunt ends, or nonoverlapping sequences.

Restriction endonucleases EcoRI and Hindlll (10 U/pL each) and their corresponding lOX incubation buffers (see Note 1). 

Connolly, B A, Eckstein, F, and Pingoud, A (1984) The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction. J. Biol Chem, 259, 10,760-10,763... 

Cutting with restriction endonucleases is very useful for moving specific pieces of DNA around from place to place. It s also a useful way to name pieces of DNA. For example, a piece of DNA that is cut from a bigger piece of DNA is often named by size and given a surname that corresponds to the two restriction enzymes that did the cutting—the 0.3-kb EcoRI-BamHI fragment. Restriction enzymes themselves are named for the bacterial strains from which they were initially isolated. 

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

The EcoRI endonuclease cleaves this hexanucleotide sequence on each strand between the G and the first A residue (reading 5 -to-30 this point of cleavage is marked by arrows in the sequences in Table 3.1. Notice that the points of cleavage are offset, or staggered, for EcoRI. Not all restriction endonucleases make staggered cuts in DNA some cleave the DNA without any offset, to produce blunt-ended cuts. 

To handle the mass of existing data, powerful computer programs have been developed and various graphical procedures have also been developed to help the human mind comprehend the results.654 655 One important problem is to define and locate what are called consensus sequences. The problem is best illustrated by examples.654 The cleavage site for the EcoRI restriction endonuclease is GAATTC. There is no ambiguity. In a DNA of random sequence this would be expected to occur by chance in about (1 / 4)6 nucleotides (4 kb). On the other hand, the Hmll restriction endonuclease cleaves within the consensus sequence GTYRAC where Y = C or T and R = A or G. 

In contrast to DNAse I, the restriction endonucleases, which are discussed in Chapter 5, Section H,2 and in Chapter 26, have precise substrate sequence specificities. Three of the best known restriction endonucleases are called EcoRI,832 - 834 an enzyme which binds... 

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... 

The cloning and manipulation of genes usually depends upon the precise cutting of DNA into discrete fragments by restriction endonucleases. Many restriction enzymes generate cohesive ends (sticky ends). Thus, EcoRI produces DNA fragments with the single-stranded "tails" shown here at the 5 -ends of the cut duplexes ... 

The array of restriction endonuclease cleavage sites which has been inserted near the N-terminus of the /3-galactosidase gene occupies positions 6231 (EcoRI site) to 6238 (centre of the HaeIII site) cf. page 134, chapter 4). A restriction map of the closely related M13mp8 is given in Appendix 8. 

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