Biology 335 - Molecular Genetics

Restriction Enzymes
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Host Restriction
Restriction Enzymes
Naming Restriction Enzymes

Restriction enzymes have obscure sounding names which are directly derived from the bacterial strains from which they were first isolated. Most of the restriction enzymes we purchase commercially have been cloned and are now isolated from engineered E. coli expressing them - they still bear their original names however. Several examples of how these enzymes are named are shown below.

 
PstI
Providencia stuartii
1st enzyme isolated

 
EcoRI
Escherichia coli
strain RY103
1st enyzme isolated

 
TaqI
Thermus aquaticus
1st enzyme isolated
HindIII
Haemophilus influenzae
strain RD
3rd enzyme isolated

 
Restriction Enzyme Classification

Restriction enzymes are classified on the basis of two fundamental properties.
First, whether the enzyme cuts at its recognition site, and second whether the restriction and modification activities are associated with the same or different complexes.

 
Type 1:
cuts at recognition site
restriction and modification activities part of the same complex.

 
Type 2:
cuts at recognition site
restriction and modification activities part of different complexes

 
Type 3:
cuts an arbitrary distance from recognition site
restriction and modification activities part of same complex.

 
Most of the enzymes we use in molecular biology are Type 2 enzymes. In the event that we might want to make use of the modification system (for instance to protect cDNAs from digestion during library construction), some type 2 modification enzymes are also commercially available.

 
Restriction Sites

Restriction endonucleases recognize specific sequences 4 to 8 base pairs long. These restriction sites are typically pallindromic - they read identically on both strands.

 
The dyad symmetry of restriction sites suggests that restriction enzymes act as dimers - each subunit interacting with one half of the site. This has been confirmed for EcoRI.
Mechanistically, restriction enzymes are believed to bind non-specifically to the sugar-phosphate backbone of the target molecule. The enzyme can then use the backbone as a track to scan the DNA for an appropriate sequence by probing for sequence-specific features in the major groove of the molecule. On finding such a site, the enzyme binds, and in the presence of Mg+2 undergoes a conformational change which kinks the helix and breaks the backbone of each strand to produce fragments with 5' phosphate and 3' hydroxyl groups.

 
Depending on where in the recognition site relative to the axis of dyad symmetry the enzyme cleaves the DNA strands, three types of ends are produced.
If the enzyme cuts at the axis, blunt ends are produced.
If the enzyme cuts to the left of the axis, 5' overhangs are produced.
If the enyzme cuts to the right of the axis, 3' overhands are produced.

 
 

While each restriction enzyme recognizes and cleaves a specific sequence, a given recognition sequence may be recognized by multiple enzymes. Enyzmes which recognize the same sequence are called isoschizomers. Recently these have been further divided into two classes - isoschizomers and neoschizomers - which cleave the DNA at the same position or different positions respectively.

 
 

Frequency of Restriction Enzyme Sites

The frequency with which restriction sites occur in a random sequence can be simply calculated if the GC content of the random sequence is known. For each nucleotide position in the restriction site, determine the frequency with which that position is occupied by the appropriate base. Then multiply the frequencies together to obtain the frequency with which the complete site is observed.

 
In a Random Sequence (50% GC)

4-mer sequence will occur
1/4 x 1/4 x 1/4 x 1/4
= 1/256 bp

6-mer sequence will occur
1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4
= 1/4096 bp

8-mer sequence will occur
1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4
= 1/65536 bp

 
If instead of 50% GC, the random sequence were 40% GC, the 4-mer would cut

2/10 x 3/10 x 3/10 x 2/10
= 1/278 bp

Similarly the 6-mer would cut

2/10 x 2/10 x 3/10 x 3/10 x 2/10 x 2/10
= 1/6944 bp

and the 8-mer would cut

2/10 x 2/10 x 2/10 x 2/10 x 2/10 x 2/10 x 2/10 x 2/10
= 1/390625 bp

Average fragment size and average frequency of restriction enzyme cutting depends on both the length and the GC bias of the recognition site as well as the GC bias of the genome being cut.

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