Restriction Enzymes

What is a RESTRICTION ENZYME?

A restriction enzyme is a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at that specific site, which is known as restriction site or target sequence.

More than 400 restriction enzymes have been isolated from the bacteria that manufacture them. In live bacteria, restriction enzymes function to defend the cell against invading viral bacteriophages. Restrictions sites in the viral genome (a "happy accident" of nature, as far as the bacteria are concerned, since they don't appear to have any specific function in the virus) are cleaved by the bacterium's restriction enzymes, fragmenting and destroying the DNA of invading bacteriophages before it can incorporate into the host's genome and take over the cell.

A bacterium is immune to its own restriction enzymes, even if it has the target sequences ordinarily targeted by them. This is because the bacterial restriction sites are highly methylated, making them unrecognizable to the restriction enzyme.

Isn't evolution fantastic?

When a restriction enzyme cleaves a restriction site, the reaction creates highly reactive "sticky ends" on the broken DNA. This is useful to the biotechnologist!

By cutting open vector DNA with the same with restriction enzymes used to cleave the target DNA, complementary "sticky ends" are created. This fosters the insertion of the target DNA into the vector:

The fragment is "glued in" with DNA ligase, which creates the phosphodiester bonds necessary to complete the sugar-phosphate backbone of the newly transgenic DNA.

Identity of Restriction Enzymes

Restriction enzymes are named for the organism from which they were first isolated. For example

EcoRI is isolated from E. coli strain RY13.
Eco refers to the genus and species (1st letter of genus; 1st two letters of specific epithet)
R is the strain of E. coli
I (Roman numeral) indicates it was the first enzyme of that type isolated from E. coli RY13.
BamHI is isolated from Bacillus amyloliquefaciens strain H
Sau3A is isolated from Staphylococcus aureas strain 3A.
And so on.

Some restriction enzymes also cut DNA to form "blunt" ends (without single-stranded tails), which also can be inserted into target DNA via the action of DNA ligase.

DNA ligase isn't picky: it can't tell the difference between foreign and host DNA (who'd figure it would ever have to?), and this enables the creation of chimeric DNA--DNA from two separate sources.

Each enzyme recognizes and cuts specific DNA sequences. For example, BamHI recognizes the double stranded sequence:

5'--GGATCC--3'
3'--CCTAGG--5'

Here's another artist's conception of how this works. (Notice the "sticky ends.")

To summarize...

Most restriction enzymes are specific to a single restriction site
Restriction sites are recognized no matter where the DNA came from
The number of cuts in an organism's DNA made by a particular restriction enzyme is determined by the number of restriction sites specific to that enzyme in that organism's DNA.
A fragment of DNA produced by a pair of adjacent cuts is called a RESTRICTION FRAGMENT.
A particular restriction enzyme will typically cut an organism's DNA in to many pieces, from several thousand to more than a million!
There is a great deal of variation in restriction sites even within a species.
Although these variations do not have phenotypic expression beyond the base sequences themselves, the variants can be considered molecular "alleles," and they can be detected with sequencing techniques.
As such, they can be used in mapping studies similar to the way true genes with known phenotypic effects can be used, but skipping the breeding steps and going straight to the molecules.
These "molecular alleles" are a type of MOLECULAR MARKER, as they can be detected and located with labeled probes.

What is a RESTRICTION ENZYME?

Identity of Restriction Enzymes

5'--GGATCC--3' 3'--CCTAGG--5'

5'--GGATCC--3'
3'--CCTAGG--5'