Type II restriction enzymes

Type II restriction enzymes have received widespread application in the cloning and sequencing of DNA molecules. Their hydrolytic activity is not ATP-depen-dent, and they do not modify DNA by methylation or other means. Most importantly, they cut DNA within or near particular nucleotide sequences that they specifically recognize. These recognition sequences are typically four or six nucleotides in length and have a twofold axis of symmetry. For example, E. coU has a restriction enzyme, coRI, that recognizes the hexanucleotide sequence GAATTC ... 

Type II site-specific deoxyribonuclease [EC 3.1.21.4], also referred to as type II restriction enzyme, catalyzes the endonucleolytic cleavage of DNA to give specific, double-stranded fragments with terminal 5 -phosphates. Magnesium ions are required as cofactors. 

Fig. 5. Schematic of the construction and function of chimeric restriction endonucleases (Chandrasegaran and Smith, 1999). (a) The type IIS restriction enzyme Fokl has a nonspecific endonuclease domain (FN) and a DNA-binding domain. Swapping Fokl s DNA binding-domain for that of another DNA binding-domain results in a chimeric restriction enzyme with new specificity. The flexibility resulting from less intimate contact between the Fn domain and its new DNA-binding domain translates into DNA cleavage at several locations near the binding site, (b) The modular nature and tunable specificity of zinc...

Rational design to extend specificities of type II restriction enzymes... 

These enzymes are extraordinarily abundant over 1200 restriction endonucleases had been isolated and characterized by early 1990. Of three classes defined, type II restriction enzymes, which generally cut within their recognition sequences, have found uses in a host of biomedical research and diagnostic applications to be discussed below. Type 1 enzymes cut nonspecifically many nucleotides distal to specific recognition sequences and contain both restriction enzyme and DNA modification (see below) activities on different subunits of multienzyme complexes. Type III restriction enzymes share the multienzyme aspeas of type I enzymes but vary in other properties such as ATPase activity and cofactor requirements. 

Type II restriction enzymes (Table 1) require physiologic pH, usually divalent cations (primarily Mg ), and, most importandy, a DNA substrate with the... 

Interestingly, transposon Tn7 behaves in a similar way but, in this case, the 5 endonuclease activity is supplied by a separate enzyme whose structure resembles that of a type II restriction enzyme (30), and cleavage occurs cleanly at the transposon tip rather than within the TE. Transposition of Tn7, like most bacterial elements, does not leave a scar. In both the Tc-Mariner transposon group and the Tn7 family of transposons, the transposase then cleaves and transfers the 3 end in a true DDE transposition reaction (Fig. le). 

A common catalytic core in type II restriction enzymes Section 9.3.4... 

Type II Restriction Enzymes Have a Catalytic Core in Common and Are Probably Related by Horizontal Gene Transfer... 

Type II restriction enzymes are prevalent in Archaea and Eubacteria. What can we tell of the evolutionary history of these enzymes Comparison of the amino acid sequences of a variety of type II restriction endonucleases did not reveal significant sequence similarity between most pairs of enzymes. However, a carefiil examination of three-dimensional structures, taking into account the location of the active sites, revealed the presence of a core structure conserved in the different enzymes. This structure includes P strands that contain the aspartate (or, in some cases, glutamate) residues forming the magnesium ion binding sites (Figure 9.44). 

These observations indicate that many type II restriction enzymes are indeed evolutionary related. Analyses of the... 

Figure 9.44 A conserved structural core in type II restriction enzymes. Four conserved structural elements, including the active-site region (in blue), are highlighted in color in these models of a single monomer from each dimeric enzyme. The positions of the amino acid sequences that form these elements within each overall sequence are represented schematically below each structure. [Drawn from IRVB.pdb lERI.pdb IBHM.pdb.]...

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... 

Type II restriction enzymes (R ENases) are a large group of DNA endonucleases that are distinguished from other R—M systems by two characteristics (i) the ENase activity for DNA restriction is physically and functionally separate from... 

This section covers the general features of type II R ENases and then more specifically EcoRI, one of the typical and most thoroughly studied type II restriction enzymes. Because the major use of type II restriction enzymes is to cut dsDNA in a site-specific manner, their specific applications are described within each appropriate subsection. 

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