EcoRI endonuclease

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. 

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... 

Dugaiczyk, A., Boyer, H. W., and Goodman, H. M. (1975). Ligation of EcoRI Endonuclease-Generated Fragments into Linear and Circular Structures. J Mol Biol 96 171. 

Lesser DR, Kurpiewski MR, Jen-Jacobson L. The energetic basis of specificity in the EcoRI endonuclease-DNA interaction. Science 1990 250 776-786. 

Jayaram J, McConnell KJH, Dixit SB, Beveridge DL. Free energy analysis of protein-DNA binding the EcoRI endonuclease-DNA complex. J Comput Phys 1999 151 333-357. 

Bobst AM, Pauly GT, Keyes RS, Bobst EV (1988) Enzymatic sequence-specific spin labeling of a DNA fragment containing the recognition sequence of ecori endonuclease. FEBS Lett 228(l) 33-36... 

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