Restriction enzymes are proteins that function as molecular scissors, cutting DNA at specific locations. Found naturally in bacteria, these enzymes serve as a defense mechanism against invading viruses by cleaving foreign DNA. They do not cut DNA indiscriminately, but instead identify highly specific sequences of nucleotides known as recognition sequences or restriction sites. This precision allows bacteria to degrade foreign genetic material without harming their own.
The names of these enzymes are derived from the bacteria they originate from; for example, EcoRI comes from Escherichia coli strain RY13. This specificity is the foundation of their use in science, providing a predictable way to manipulate DNA.
Defining Features of Recognition Sequences
Recognition sequences are defined by their specific nucleotide order and length, consisting of 4 to 8 base pairs. The length of the sequence directly impacts how frequently it appears in a genome by chance. A shorter 4-base sequence occurs more often than a longer 8-base sequence, making enzymes that recognize longer sites more specific for cutting larger genomes into fewer pieces.
A defining characteristic of most recognition sequences is that they are palindromic. This means the sequence on one DNA strand reads the same in the 5′ to 3′ direction as the sequence on the complementary strand read in its 5′ to 3′ direction. For instance, the enzyme EcoRI recognizes the sequence 5′-GAATTC-3′, and its complementary strand is 3′-CTTAAG-5′, which, when read from 5′ to 3′, is also GAATTC.
This palindromic nature is tied to the structure of the enzymes, which often bind to the DNA as a dimer, a complex of two identical protein subunits. The sequence itself is a string of the four nucleotide bases: adenine (A), guanine (G), cytosine (C), and thymine (T). While most enzymes are highly specific to a single sequence, some can recognize a family of similar sequences.
How Restriction Enzymes Interact with Recognition Sequences
The interaction between a restriction enzyme and its recognition sequence involves the enzyme physically binding to and cutting the DNA. The enzyme scans a DNA molecule until it encounters its target sequence. Upon recognition, the enzyme binds to the DNA and initiates a process that breaks the phosphodiester bonds that form the backbone of the molecule, resulting in cleavage.
The cut can occur in two distinct ways. A “sticky end,” or cohesive end, is created when the enzyme cuts the two DNA strands asymmetrically. This leaves short, single-stranded overhangs, such as the AATT overhangs produced by EcoRI. These overhangs are “sticky” because they can easily form hydrogen bonds with complementary overhangs.
Conversely, a “blunt end” results when the enzyme cuts symmetrically, cleaving both strands at the exact same point. This leaves no single-stranded overhang, and while blunt ends can be joined, the process is less efficient than with sticky ends. Most restriction enzymes used in molecular biology are Type II enzymes, which are useful because they cut precisely within or very near their recognition site.
Factors Affecting Recognition and Cleavage
Several factors can influence an enzyme’s ability to recognize and cleave a DNA sequence. One is DNA methylation, a process where a methyl group (–CH3) is added to certain nucleotide bases. In bacteria, this modification is a self-protection system. The bacterium’s own DNA is methylated at recognition sites, which blocks its restriction enzymes from cutting it, while unmethylated foreign DNA remains vulnerable.
In a laboratory setting, if the target DNA is methylated at a recognition site, the enzyme may be unable to bind or cut. This is a consideration when working with DNA from organisms that use methylation for gene regulation. Scientists must choose enzymes that are insensitive to the specific methylation pattern of their DNA sample.
Another factor is “star activity,” the relaxation of an enzyme’s specificity, causing it to cleave sequences similar but not identical to its defined recognition site. This undesirable artifact occurs under non-optimal reaction conditions such as incorrect buffer pH or salt concentration, prolonged incubation times, or high concentrations of the enzyme or glycerol. This loss of precision can lead to unintended cuts in the DNA, complicating experimental results.
The Role of Recognition Sequences in Biotechnology
The precise nature of restriction enzyme recognition sequences enables numerous biotechnology applications. The ability to cut DNA at predictable locations allows scientists to isolate, modify, and recombine genetic material with great accuracy, which has revolutionized fields from medicine to agriculture.
A primary application is gene cloning. To insert a specific gene into a bacterial plasmid (a small, circular DNA molecule), scientists use the same restriction enzyme to cut both the gene and the plasmid. This creates compatible sticky ends on both pieces of DNA, allowing the gene to be inserted into the plasmid and joined by another enzyme called DNA ligase. The result is a molecule of recombinant DNA.
These sequences are also used for DNA mapping and genetic fingerprinting. Restriction mapping involves cutting DNA with various restriction enzymes to create a map of their recognition sites. Another technique, Restriction Fragment Length Polymorphism (RFLP) analysis, exploits variations in DNA sequences between individuals. Differences in the presence or absence of a recognition site result in DNA fragments of different lengths after digestion, creating a unique pattern for identification or to diagnose genetic disorders.