Some restriction endonucleases

Fig. 1. Nucleotide sequence of the SFV 26 S RNA (top row), the corresponding amino acid sequence (middle row), and the amino acid sequence of the Sindbis virus structural proteins (bottom row). Nucleotides are numbered from the 5 end of the RNA molecule and all amino adds from the amino terminus of each protein. The amino- and the carboxyl-terminal ends of each protein are indicated hy arrows, glycosylation sites by triangles, and membrane-spanning regions of the viral glycoproteins by underlines for Sindbis virus and overlines for SFV. Amino acids in boxes are negatively charged (Asp and Glu), and those circled are positively charged (Lys and Arg). Some restriction endonuclease cleavage sites are shown on the nucleotide sequence. The alignment of the amino acid...

Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end. These unpaired strands are referred to as sticky ends (Fig. 9-3a), because they can base-pair with each other or with complementary sticky ends of other DNA fragments. Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called blunt ends (Fig. 9-3b). 

Fig. 1.4. Specificities of some restriction endonucleases. The two fold axis of symmetry is indicated by the dotted line and the arrows indicate the cleavage sites. Note that in (i) and (ii) the products possess 5-single stranded extensions of four nucleotides in (iii) and (v) the products are flush (blunt) ended and in (iv) and (vi) the products have 3 -single stranded extensions.

Figure 1. Some restriction endonucleases hydrolysis sites on...

Figure 6.1. Specificities of Some Restriction Endonucleases. The base-pair sequences that are recognized by these enzymes contain a twofold axis of symmetry. The two strands in these regions are related by a 180-degree rotation about the axis marked by the green symbol. The cleavage sites are denoted by red arrows. The abbreviated name of each restriction enzyme is given at the right of the sequence that it recognizes.

Some Restriction Endonucleases and Their Cleavage Sites ... 

Fig. 1 Some restriction endonuclease cleavage sites in SV40 DNA.

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). 

Zahner, V. Priest, F. G. Distribution of restriction endonucleases among some ento-mopathogenic strains of Bacillus sphaericus. Letts. Appl. Microbiol. 1997, 24, 483-487. 

Restriction endonucleases are at the core of recombinant DNA technology. The specificity of some of these enzymes is described in Chapter 13. Fortunately the restriction endo-nucleases cleave DNA at... 

Figure 12.10 Some genes and restriction sites on the E. coli plasma pBR322. Eco RI, Pst I, and Sal I are restriction endonucleases and the sites cleaved by these enzymes.

DNA molecules, whether linear or circular, can be cut by means of enzymes known as restriction enzymes. A restriction enzyme is an enzyme that recognizes certain sequences of nitrogen bases and breaks the bonds at some point within that sequence. Since these enzymes work on bonds within the DNA molecule, they are sometimes called restriction endonucleases. 

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. 

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... 

The cuts in the two strands are made at the points indicated by the arrows. This one endonuclease will cut almost any DNA into long pieces averaging about 5000 base pairs each. These pieces can in turn be cleaved by other restriction endonucleases to form smaller fragments. Since there are about 2400 of these enzymes known, with 188 different specificities,536 it is possible to cut any piece of DNA down to a size of 100-500 base pairs, ideal for sequencing.537 539 Each fragment has known sequences at the two ends. Some restriction enzymes cleave outside their specific recognition sequence (see Table 26-2). Some recognize 16-nucleotide palindromes and cut at rare sites. 

A multitude of nucleases cleave DNA, single- or double-stranded. They range from the pancreatic digestive enzyme DNase I through specialized nucleases that function during DNA repair and the hundreds of restriction endonucleases that have become so valuable in modern laboratory work. Some nucleases leave a 3 -phosphate ester at a cut end in a DNA chain, while others leave a 5 -phosphate end.824 Many nucleases are dealt with in later chapters. Only a few will be mentioned here. 

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