Humans get all the credit for scientific discoveries perhaps because we’re the ones with the capacity to think and act on our ideas. Enzymes, on the other hand, are just molecules that catalyze chemical reactions and don’t have the same capacity for thought. But, if enzymes could think and talk, I’m betting they would have interesting things to say about the discoveries that we’ve made together, their role in those discoveries, and their hopes for a little acknowledgement. So here’s a long overdue shoutout to three not to be forgotten restriction enzymes.
Restriction enzymes, also known as restriction endonucleases, are molecules found in bacteria and archaea that cleave DNA at specific sites. These enzymes are used by bacteria as a defense mechanism to protect against invading viruses but have been harnessed by scientists for DNA mapping, gene experiments, and recombinant DNA technologies. Their many applications in biology, genetics, and biotechnology have earned them the nickname ‘the workhorse of the lab’. While there are ~650 commercially available restriction enzymes and over 4000 studied restriction enzymes we’ll focus here on three of the most used and most influential ones – EcoRI, HindIII, and BamHI.
EcoRI, pronounced “eco R one”, is a homodimer which means it is formed by two identical proteins. This enzyme wraps around double-stranded DNA and catalyzes DNA’s cleavage creating predictable fragments of DNA and “sticky” cut sites. This restriction enzyme has a recognition site of 5’-GAATTC-3’ and it cleaves at 5’-AATTC-3’. EcoRI was instrumental in the first recombination DNA experiment. In the spring of 1973, biologists Herbert Boyer and Stanley Cohen cleaved a bacteria plasmid (pSC1010) using EcoRI and mixed the linearized plasmid with similarly cut DNA containing a gene that gave bacteria immunity to tetracycline. They then treated the mixture with an additional enzyme that enabled the plasmid and cut DNA ends to join creating a new recombinant plasmid. When this plasmid was reintroduced to calcium-chloride-treated bacteria, the bacteria were able to grow on tetracycline-containing agar. EcoRI was also used extensively in the human genome project. By helping researchers map the entire human genome the enzyme has furthered our understanding of the genetic basis of diseases and also what makes us human. EcoRI has also been used in many gene knockout experiments which has helped humans to identify the cascading effects of specific genes.
HindIII, pronounced “hin D three”, was first discovered in the bacteria Haemophilus influenzae. But while the bacteria is associated with a range of human infections, this restriction enzyme has been a biomedical force for good. HindIII is commonly used in the pharmaceutical industry for the production of biopharmaceuticals, such as monoclonal antibodies, recombinant proteins, and DNA vaccines. In addition, the enzyme has also been used to produce gene base diagnostics and gene editing therapies. Much like EcoRI, HindIII cuts double-stranded DNA to produce predictable DNA fragments and sticky ends. This restriction enzyme has a recognition site of 5’-AAGCTT-3’, and it cleaves at the sequence 5’-AGCTT-3’. Unlike EcoRI and most other type II restriction enzymes, HindIII has catalytic activity only in the presence of Mg2+ and performs poorly when this ion is substituted for other cofactors such as Mn2+. Another feature that sets this restriction enzyme apart is that, despite years of heavy use, scientists still don’t completely understand how HindIII cleaves DNA. In spite of or perhaps because of the mystery, HindIII is a still a much beloved research tool.
BamHI, pronounce Bam H one, has a name right out of a comic strip and a superpower to match – it can recognize short sequences of six DNA base pairs and cut through the DNA at this specific site to create fragmented DNA with sticky ends. The recognition sequence for BamHI is 5’-GGATCC-3’ and it cleaves the sequence 5’-GATCC-3’. BamHI is one of the most commonly used restriction enzymes because of its relative stability in a variety of buffers and a wide range of temperatures. This means that BamHI is often used in combination with other restriction enzymes. The enzyme was discovered in 1977 by Dr. Richard Roberts, Dr, Gary Wilson, and Dr. Frank Young who published their finding in this Nature article. Today the enzyme is still used in cloning, diagnostics, and genetic therapy and is also a popular model to study enzymatic reactions.
Ready to learn more about these powerful enzyme partners?
- Check out our other posts about restriction enzymes.
- Type in your choice of restriction enzyme in the REBASE database and discover everything from its target sequence to its crystalline structure to recent publications involving the enzyme.
- Get your students co-experimenting with these amazing molecules with one of our experiments.