In the early 1950s,experiments by two teams of researchers, Salvador Luria working with Mary Humanand Joe Bertani working with Jean Weigle, showed that some strains of bacteriawere more resistant to viral infections than others. Viruses that infectbacterial cells are called bacteriophages. Their main goal is to produce morebacteriophages by injecting their genome into a bacterial host cell, using thehost cell machinery to copy their genome, and expressing bacteriophage genes.The researchers found, however, that some strains of bacteria appeared to beless vulnerable to bacteriophage infections than others and resisted thehijacking of their cell machinery by bacteriophages. A deeper look into the apparentself-defense mechanisms of these bacteriophage-resistant bacteria revealedtheir secret weapon: a group of enzymes called restriction endonucleases, orrestriction enzymes. These enzymes opened the path to a powerful research toolthat scientists later used not only to sequence genomes, but also to create thefirst synthetic cell, two scientific research milestones that affect us all insome way.
The discovery of restrictionenzymes began with a hypothesis. In the 1960s, Werner Arber observed a dramaticchange in the bacteriophage DNA after it invaded these resistant strains ofbacteria: It was degraded and cut into pieces. In an attempt to explain theresistance of certain bacterial strains to bacteriophage infection, Arber thenposited that bacteriophage-resistant bacterial cells might express a specificenzyme that degrades only invading bacteriophage DNA, but not their own DNA. How,though, would a DNA-degrading enzyme distinguish between the two? Arberhypothesized that bacterial cells might express two types of enzymes: arestriction enzyme that recognizes and cuts up the foreign bacteriophage DNAand a modification enzyme that recognizes and modifies the bacterial DNA toprotect it from the DNA-degrading activity of its very own restriction enzyme.He predicted that the restriction enzyme and the modification enzyme act on thesame DNA sequence, called a recognition sequence. In this way, the bacterialcell's own self-defense mechanism, which aggressively degrades invadingbacteriophage DNA, would also protectits own DNA from degradation at the same time. This prediction was confirmed inthe late 1960s by Stuart Linn and Arber when they isolated a modificationenzyme called methylase and a restriction enzyme responsible for bacteriophageresistance in the bacterium Escherichiacoli. The methylase enzyme added protective methyl groups to DNA, and therestriction enzyme cut unmethylated (unprotected) DNA at multiple locationsalong its length.
A few years later, in 1970,Hamilton Smith not only independently verified Arber's hypothesis, but alsoelaborated on the initial discovery by Linn and Arber. He successfully purifieda restriction enzyme from another bacterium, Haemophilus influenzae (H.influenzae), and definitively showed that this enzyme cut DNA in the centerof a specific six-base-pair sequence. Interestingly, he also showed that thisenzyme did not cut at this very sameDNA sequence when it occurred in H.influenzae host cell DNA. Building on this result, a first glimpse of howrestriction enzymes could be useful tools for scientific research emerged oneyear later in experiments carried out by Dan Nathans and Kathleen Danna. Theyused Smith's restriction enzyme to cut the 5,000 base-pair genome of the SV40virus, which infects monkey and human cells, and identified eleven differentlysized pieces of DNA. Nathans's lab later showed that when the SV40 genome wasdigested with different combinations of restriction enzymes, the sizes of theresulting pieces of DNA could be used to deduce a physical map of the SV40viral genome, a groundbreaking method for inferring gene sequence information.This method of cutting a DNA molecule into smaller pieces is called arestriction enzyme digest, and it quickly became a powerful tool for generatingphysical maps of a multitude of genomes, which at the time was a preciousrevelation in the early stages of genome sequencing. For this groundbreakingset of discoveries, Arber, Smith, and Nathans were jointly awarded the NobelPrize in Physiology or Medicine in 1978.
Given the vast geneticdiversity among bacteria, it follows that different bacterial strains expressdifferent restriction enzymes, allowing them to balance their own genes againstthose of invading bacteriophages. The known variety of restriction enzymes isstaggering: To date, more than 4,000 different restriction enzymes thatcollectively recognize more than 360 different recognition sequences have beenisolated from a wide variety of bacterial strains. Based on DNA sequenceanalysis, scientists know that there are many more restriction enzymes outthere waiting to be characterized. The recognition sequences of these enzymesare typically four to six base pairs in length, and they are usuallypalindromic, which means that their recognition sequence reads the same in the5' to 3' direction on both DNAstrands. There are four different categories of restriction enzymes. Type Irestriction enzymes cut DNA at random locations far from their recognitionsequence, type II cut within or close to their recognition sequence, type III cutoutside of their recognition sequence, and type IV typically recognize amodified recognition sequence.
Type II restriction enzymes,which cut within their recognition sequence, are the most useful for laboratoryexperiments. Scientists use them to cut DNA molecules at interesting specificlocations and then reattach different DNA sequences to each other using anenzyme called DNA ligase, creating new, recombined DNA sequences, or essentiallynew DNA molecules. This powerful approach to cutting and pasting DNA moleculesis known as DNA cloning or recombinant DNA technology. When they act on a DNAmolecule, restriction enzymes produce "blunt" ends when they cut in the middleof the recognition sequence, and they yield "sticky" ends when they cut at therecognition sequence in a staggered manner, leaving a 5' or 3' single-stranded DNAoverhang. Any two blunt ends can be joined together, but only sticky ends withcomplementary overhangs can be connected to each other. Restriction enzymedigestion continues to be one of the most common techniques used by researcherswho carry out DNA cloning experiments.
Today, researchers rely onrestriction enzymes to perform virtually any process that involvesmanipulating, analyzing, and creating new combinations of DNA sequences. Amongthe many new combinations are DNA cloning, hereditary disease diagnosis,paternity testing, forensics, genomics (e.g., the human genome project), epigenetics,genetically modified organisms, and biotechnology. Indeed, without thediscovery of restriction enzymes, the fields of recombinant DNA technology,biotechnology, and genomics as we know them today would not exist. In 2010,forty years after he purified the first restriction enzyme, Smith was part ofthe research team that used these very enzymes to build the first syntheticbacterial cell. Led by Craig Venter, this team of scientists used machines tochemically synthesize the one million base-pair Mycoplasma mycoides (M.mycoides) bacterial genome in 1,080 base-pair pieces that were then joinedtogether to form a complete synthetic genome. Along the way, Venter and hiscolleagues used restriction enzymes to help clone and analyze the syntheticgenome. In the final step, they transplanted the synthetic M. mycoides genome into a Mycoplasmacapricolum bacterial cell and showed that recipient cells harboring onlythe synthetic M. mycoides genome werecapable of reproducing and exhibited characteristics of M. mycoides cells. In this Spotlight, you'll find a broad range ofresources to help you gain a deeper understanding of how restriction enzymes affectedthe field of molecular biology and our ability to manipulate DNA, as well ashow they continue to serve as an invaluable tool for research scientists.
--Heidi Chial, Ph.D. (BioMed Bridge, LLC)
