Restriction Enzymes

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Restriction enzymes (also known as restriction endonucleases) are enzymes that cut double-stranded DNA at very specific recognition sites. They were originally discovered in bacteria that use them to restrict the growth of viruses but are now among the workhorse enzymes of biotechnology and recombinant DNA research.

It has long been known that bacteria are susceptible to attack by viruses known as bacteriophages ("eaters of bacteria"). The presence of restriction enzymes in bacteria is part of the defense system against bacteriophages that has evolved in these bacteria. These highly specific enzymes will scan DNA until a certain sequence of nucleotide bases is identified. The specificity is such that the sequence is apt to occur at only one or two sites in the viral DNA, with no such occurrence in the host bacterial DNA. Restriction enzymes recognize a sequence such as:

5′ G A A T T C 3′

3′ C T T A A G 5′

Interestingly, the enzyme can recognize the paired sequences from either strand because they are a palindrome (reading the same in either direction). The restriction enzyme can cut this palindromic sequence in one of two manners: across both strands at the same spot, or in a staggered manner that yields free single-stranded ends called "sticky ends." These sticky ends have proved most useful in recombinant DNA work.

The names of restriction enzymes are derived from their bacterial sources. One of the enzymes most widely used in recombinant DNA work is Eco R1, which is isolated from Escherichia coli RY13. Other examples include HindII (isolated from Haemophilus influenza Rd), and Xba I (isolated from Xanthomonas badrii ). The specificity of each enzyme allows researchers to cut DNA in a predictable and reproducible manner. Using Eco R1 on the above sequence, one would always obtain the ends:

————– 5′ G     A A T T C 3′ ————–

————– 3′ C T T A A      G 5′ ————–

A small circular piece of DNA (such as a bacterial plasmid) with one Eco R1 site would yield a linear piece of DNA with the CTTAA sticky ends. Now suppose one could obtain the DNA (the gene) coding for a foreign protein such as human insulin. At each end of the insulin gene, one could attach complementary single-stranded sticky ends that would exactly "match" the ends of the cut plasmid. As if made of Velcro, the complementary ends would stick together, and if one were to use an enzyme called DNA ligase to form a stronger covalent bond between parts joined together, one would have incorporated a human (or other species) gene into bacterial DNA. This technique is known as recombinant DNA. The bacteria can now be grown in large batches and made to synthesize the foreign protein (insulin).

SEE ALSO Deoxyribonucleic Acid ; Enzyme ; Recombinant Dna .

C. Larry Bering

Bibliography

Voet, Donald, and Voet, Judith G. (1995). Biochemistry, 2nd edition New York: John Wiley.

Internet Resources

Peters, Pamela. "Restriction Enzymes Background Paper." Access Excellence Activities Exchange. Available from http://www.accessexcellence.org .