Restriction endonucleases Type

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

Pingoud, A., Fuxreiter, M., Pingoud, V. and Wende, W. (2005) Type II restriction endonucleases structure and mechanism, Cell.Mol.Life Sci. 62, 685-707. 

This activity is intended to be performed in conjunction with Experiment 66. Restriction endonucleases, or restriction enzymes, cleave DNA at specific base sequences, fragmenting the DNA into smaller pieces. The two strands of a DNA double helix are cleaved at different places, resulting in uneven fragments called sticky ends. Cleavage of DNA by restriction enzymes is a required first step in various types of DNA analysis, including DNA fingerprinting and recombinant DNA technology. 

At least one type of palindrome recognized by a restriction endonuclease... 

Site-specific deoxyribonuclease (type 11)— restriction endonuclease ... 

The recognition sequences of type II restriction endonucleases are usually symmetric they have a twofold axis of symmetry so that they read the same in the 5 -to-3 sense along opposite—but, of course, complementary—strands. Such sequences are... 

There are three types of restriction endonucleases, designated I, II, and III. Types I and HI are generally large, multisubunit complexes containing both the endonucle-... 

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. 

TABLE 9-2 Recognition Sequences for Some Type II Restriction Endonucleases... 

Key enzymes in gene cloning include restriction endonucleases (especially the type II enzymes) and DNA ligase. 

A restriction enzyme is named according to the organism from which it was isolated. The first letter of the name is from the genus of the bacterium. The next two letters are from the name of the species. An additional subscript letter indicates the type or strain, and a final number is appended to indicate the order in which the enzyme was discovered in that particular organism. For example, Haelll is the third restriction endonuclease isolated from the bacterium Haemophilus aeqyptius. 

The type II restriction endonucleases, which are the ones most widely used in molecular biology, are relatively small 50- to 100-kDa monomeric or dimeric proteins. About 2400 different enzymes with 188 different specificities had been isolated by 1995.83/84 The sites of attack, in most instances, are nucleotide sequences with a twofold axis of local symmetry.85 For example, the following sites of cleavage have been identified for two restriction endonucleases encoded by the DNA of R-factor plasmids of E. coli and for a restriction enzyme from Hemophilus influenzae. In the diagrams i are sites of cleavage, are sites of methyla-tion, and are local twofold axes (centers of palindromes) N can be any nucleotide with a proper base pairing partner. 

Parental template DNA (plasmid DNA or short DNA amplified by PCR sequence or excised by restriction endonucleases) can be a problematic contaminant leading to the production of either wild-type clones or other false recombination products. If this happens, remove the parent DNA. There are several ways of achieving this ... 

---