2 Enzymatic Modification of DNA and RNA
27
ism of origin (three initialletters, e.g. Hin), the bacterial strain (one letter,
e.g. d), and by roman numerals (e.g. II) if more than one enzyme has been
isolated from a bacterium. More than 150 different cleavage sites have been
found. Table 1 contains a list of commercially available, frequently used
type II restriction endonucleases.
Recognition sequence
The recognition sequences are usually 4 to 8 bp long and frequently display
twofold symmetry. They represent palindromic sequences, i.e. they have the
same sequence when read forward on one DNA strand and backward on the
complementary strand. A given 4 bp site is calculated to occur every 4 4 = 256
bp, a 6 bp site every 4 6 = 4096 bp, and an 8 bp site every 4 8 = 65536 bp. The site
distribution in native DNA, however, is nonrandom and depends in part on
the base composition, so that a specific DNA region may be cut more or less
frequently than the statistical average. For example, Notl has an 8 bp recognition sequence which includes CpG dinucleotides that occur very infrequently in mammalian DNA. It produces fragments of 1 to 1.5 million bp
("rare cutter") and is therefore used for long range physical mapping of
DNA.
lsoschizomers and compatible ends
Several enzymes isolated from different organisms have identical recognition sequences. These enzymes are called isoschizomers. As shown in Figure
3, identical recognition sequences can be cleaved differently by isoschizomers. Cleavage ofthe recognition sequence GGTACC by Asp718, and Kpnl,
generates two fragments that cannot be joined ("ligated") together. Enzymes may have different recognition sites but produce identical or compatible ends. In this example, DNA fragments cleaved by Mbol and BamHI,
could be directly ligated together.
Mechanism
How do restriction endonucleases achieve their high specificity? The mechanism of action has been studied extensively for EcoRI (McClarin et al.
1986). It has been shown that the symmetry of the palindromic target sequence ( GAATTC) is matched by the symmetry of the enzyme. The enzyme
27
ism of origin (three initialletters, e.g. Hin), the bacterial strain (one letter,
e.g. d), and by roman numerals (e.g. II) if more than one enzyme has been
isolated from a bacterium. More than 150 different cleavage sites have been
found. Table 1 contains a list of commercially available, frequently used
type II restriction endonucleases.
Recognition sequence
The recognition sequences are usually 4 to 8 bp long and frequently display
twofold symmetry. They represent palindromic sequences, i.e. they have the
same sequence when read forward on one DNA strand and backward on the
complementary strand. A given 4 bp site is calculated to occur every 4 4 = 256
bp, a 6 bp site every 4 6 = 4096 bp, and an 8 bp site every 4 8 = 65536 bp. The site
distribution in native DNA, however, is nonrandom and depends in part on
the base composition, so that a specific DNA region may be cut more or less
frequently than the statistical average. For example, Notl has an 8 bp recognition sequence which includes CpG dinucleotides that occur very infrequently in mammalian DNA. It produces fragments of 1 to 1.5 million bp
("rare cutter") and is therefore used for long range physical mapping of
DNA.
lsoschizomers and compatible ends
Several enzymes isolated from different organisms have identical recognition sequences. These enzymes are called isoschizomers. As shown in Figure
3, identical recognition sequences can be cleaved differently by isoschizomers. Cleavage ofthe recognition sequence GGTACC by Asp718, and Kpnl,
generates two fragments that cannot be joined ("ligated") together. Enzymes may have different recognition sites but produce identical or compatible ends. In this example, DNA fragments cleaved by Mbol and BamHI,
could be directly ligated together.
Mechanism
How do restriction endonucleases achieve their high specificity? The mechanism of action has been studied extensively for EcoRI (McClarin et al.
1986). It has been shown that the symmetry of the palindromic target sequence ( GAATTC) is matched by the symmetry of the enzyme. The enzyme