Restriction recognition site

Figure 7.8 Sequence-specific recognition sites in the major groove of DNA for three restriction enzymes—Eco RI, Bal I, and Sma I. The DNA sequences that are recognized by these enzymes ate represented by tbe color code defined in Figure 7.7.

Only a rather limited number of base pairs is needed to provide unique and discriminatory recognition sites in the major groove. This is illustrated in Figure 7.8, which gives the color codes for the hexanucleotide recognition sites of three different restriction enzymes—Eco Rl, Bal 1, and Sma 1. It is clear that these patterns are quite different, and each can be uniquely recognized by specific protein-DNA interactions. 

Table 11.5 lists many of the commonly used restriction endonucleases and their recognition sites. Because these sites all have twofold symmetry, only the sequence on one strand needs to be designated. 

FIGURE 13.3 Restriction endonuclease Ec691 cleaves double-stranded DNA. The recognition site for is the hexameric sequence GAATTC ... 

Most useful restriction enzymes cut DNA at specific recognition sites, usually four to six nucleotides in length. There can be multiple restriction sites for a single endonuclease within a given piece of DNA, there can be only one (a unique restriction site), or there can be none. It all depends on the sequence of the specific piece of DNA in question. 

Table A4.1 presents the origins and sequence recognition sites for the restriction endonucleases in the plasmid vector.

Mutations in restriction sites (the recognition sites for restriction endonucleases such asMsfll)... 

The sequences of some recognition sites for some restriction enzymes are given in Table 3.3. 

Balendiran, K., Bonventre, J., Knott, R., Jack, W., Benner, J., SchUdkraut, 1. and Anderson, J. E. (1994). Expression, purification, and crystallization of restriction endonuclease Pvull with DNA containing its recognition site. Proteins 19,77-79. 

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... 

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