Nucleic Acids in Chemistry and Biology

Type I consists of a large enzyme complex containing subunits encoding endonuclease, methylase and
several other activities. The recognition sequence comprises a trinucleotide and a tetranucleotide separated
by about six non-specific base pairs (Table 5.2), but the endonucleolytic cleavage site can be up to 7000
base pairs distant.
Type II systems have independent endonucleases and methylases that act on the same DNA sequence.
These sequences are generally palindromic(i.e.they have a twofold axis of symmetry) and the cleavage sites
are usually within or very close to the recognition sites (Section 10.5.1). In some cases, the symmetrical
recognition sequence is interrupted (e.g. BglI), while a few enzymes recognise an asymmetric sequence and
cleave at a defined distance (e.g. MnlI). Restriction enzymes cleave both strands of the DNA either symmet-
rically to give blunt endsor asymmetrically to give sticky ends. A vast range of enzymes with different speci-
ficities has been isolated from a wide variety of organisms and type II restriction enzymes are highly
useful tools in recombinant DNA research, and the products of cleavage (restriction fragments) can often
be rejoined using DNA ligase (Section 5.3.5).
The type III system shares features in common with both type I and type II. There are two independent
polypeptides, one of which acts independently as a methylase, but both are required for specific endonucle-
olytic activity. In the case of EcoPI, for example, the recognition sequence is an asymmetric pentanucleotide
and the cleavage site is 25 bp downstream (Table 5.2).

5.3.2 Other Nucleases

Almost every organism contains a wide variety of nucleases, of which some are involved in the salvage of
nucleotides and some feature as intrinsic activities of proteins used in replication and repair processes.
Apart from non-specific nucleases, such as DNase I, and ribonucleases, there are several other nucleases
that are used in the manipulation of DNA and RNA (Table 5.3).

Nucleic Acids in Biotechnology 175

Table 5.2 Some restriction endonucleases and their recognition

sequences. N signifies any nucleotide. Cleavage sites

indicated
 

Type Enzyme Recognition site

Type I EcoK A A C (N) 6 G T A C

T T G (N) 6 C A C G

EcoB T G A (N) 8 T G C T

A C T (N) 8 A C G A

Type II EcoRI G 
 A A T T C

C T T A A  G

SmaI C C C 
 G G G

G G G  C C C

PstI C T G C A 
 G

G  A C G T C

Sau3A1 
 G A T C

C T A G 

NotI G C 
 G G C C G C

C G C C G G  C G

BglI G C C N N N N 
N G G C

C G G N  N N N N C C G

MnlI C C T C (N) 7 


G G A G (N) 7 

Type III EcoPI A G A C C

T C T G G