What are you going to learn?
- What are restriction enzymes?
- How do restriction enzymes work?
- Why are restriction enzymes so useful?
- What is recombinant DNA technology?
- terms: cohesive (sticky) ends, recognition sequence, restriction site, blunt-ended fragments, engineered nucleases, recombinant DNA technology, zinc-finger nuclease (ZFNs), transcription aktivátor-like effector nuclease (TALEN)
Restriction enzymes (or restriction endonucleases) are enzymes produced by bacteria that recognize and cut specific nucleotide sequences in DNA, which we call recognition sequences or restriction sites. The enzymes cut both strands of DNA in the site by cleaving the phosphodiester bonds (the bonds between a phosphate group and a deoxyribose). They recognize short sequences of usually 4 to 6 base pairs. These recognition sequences are usually palindromic, which means that they read the same on the two complementary DNA strands in the 5’-to-3’ direction.
When restriction enzymes cut DNA, some of them make staggered cuts. This way we get the so-called cohesive or sticky ends that are complementary to each other and can pair (anneal) with another complementary fragment cut by the same enzyme forming hydrogen bonds between the complementary base pairs. DNA ligase can then join the fragments together. Restriction enzymes can also cut in the middle of its recognition sequence producing blunt-ended fragments.
The problem with restriction enzymes described above is that their recognition sequences are very short, and they therefore produce thousands of DNA fragments. For that reason, geneticists have designed engineered nuclease that recognize longer DNA sequences. Engineered nucleases consist of a restriction enzyme and a protein that is responsible for binding to a specific DNA sequence. An example of engineered nucleases are zinc-finger nucleases (ZFNs), which use a DNA-binding domain called a zinc finger and a restriction enzyme, or a transcription activator-like effector nuclease (TALEN), which contains a protein that binds to sequences in promoters.
Restriction enzymes are useful when we want to cut DNA into fragments. It is important to say that each enzyme always cuts a DNA molecule at the same site. For these reasons, they are used in recombinant DNA technology, molecular techniques used for locating, isolating, and altering DNA segments (recombinant means that it combines DNA that comes from different sources).
References:
Klug, W. S., Cummings, M. R., Spencer, C. A., Palladino, M. A., & Killian, D. (2019). Concepts of Genetics. Pearson.
Pierce, B. A. (2019). Genetics: A Conceptual Approach (Seventh ed.). W. H. Freeman.