Classic Experiment 9.2 DEMONSTATING SEQUENCE-SPECIFIC CLEAVAGE BY A RESTRICTION ENZYME B acteria exhibit a phenomena, known as host restriction, whereby they can both recognize and cleave foreign DNA, preventing it from interfering with the bacterial life cycle. By purifying and characterizing one of the enzymes involved in host restriction, Hamilton Smith gave molecular biology one of its most important tools, an enzyme that cleaves DNA at a specific sequence. Background At the time of Hamilton Smith’s work, host restriction was a well-characterized, yet highly intriguing phenomenon. It was well known that DNA from one species of bacteria could not be used to transform a second species of bacteria. When researchers simply mixed DNA from one bacteria with a lysate from a second bacterial species, the DNA was cleaved. The bacteria had evolved a system to recognize and cleave foreign DNA. In 1965, Werner Arber hypothesized that bacteria must produce an enzyme capable of recognizing and cleaving foreign DNA at specific sequences. How did a bacterium determine which DNA was foreign, and which was its own? It seemed unlikely that a bacterium could exclude specific sequences in its genome, from the action of this nuclease. More likely, a bacterium somehow modified its own DNA at these sequences, so it could be spared from cleavage. The existence of a second enzyme was thus hypothesized, one that could modify the DNA by methylation at the site where cleavage occurred, thereby preventing cleavage by the sequence-specific nuclease. With these hypotheses in hand, the hunt for the enzymes could begin. In 1968, Mathew Meselson reported the purification from E. coli of one of these enzymes now called restriction enzymes or restriction endonucleases. Although the E. coli enzyme catalyzed the cleavage of non-E.coli DNA, Meselson could not demonstrate that this cleavage was sequence specific. In fact, proving that these bacterial enzymes cleave DNA at a specific sequence would be a tricky manner, as this research was conducted before the advent of the relatively simple DNA-sequencing techniques now available. Following on Messelson’s work, Smith set out to purify a second restriction enzyme, this time from H. influenzae, and to demonstrate that it does indeed cleave DNA in a sequence-specific manner. The Experiment The first step in the successful purification of a new enzyme is devising an assay that measures the known activity of the enzyme as it is being purified. The activity of a restriction enzyme is to catalyze the cleavage of foreign DNA, so this was the logical activity to monitor. To do so, Smith took advantage of the fact that genomic DNA from bacteria is quite viscous, however as nucleases begin to degrade the bacterial DNA, its the overall viscosity decreases. Therefore, Smith could monitor the purification of his restriction enzyme by measuring the decrease in viscosity of a foreign DNA after treatment with a sample of the protein after each step in the purification scheme. Smith mixed cell extracts of H. influenzae with intact DNA from either H. influenzae or the Salmonella bacteriophage P22. Using a device called a viscometer, he measured how the DNA from P22 became less viscous over time, while the H. influenzae DNA displayed no change in viscosity. This would be the assay he would use throughout the purification scheme. Smith used a variety of established methods to separate bacterial lysates into smaller pools of proteins. Each method separated the lysate based on a different physical property of the proteins (and other biomolecules) that make up the lysate. This allowed the lysate to be divided into subsamples known as fractions. After each step in the purification, every fraction was separately assayed for the ability to cleave P22 DNA. Fractions that contained the enzyme activity were subjected to yet another purification method, and the process was continued until a pure enzyme was obtained. Smith called the purified restriction enzyme endonuclease R. Next Smith determined some of the basic characteristics of endonuclease R. He used endonuclease R to digest DNA from the bacteriophage T7, then estimated the number of sites where the DNA was cleaved. He discovered that endonuclease R did not completely degrade T7 DNA, but rather cleaved it at approximately 40 sites. Since T7 DNA contains approximately 40,000 bases, cleavage occurred at only 0.1 percent of the possible sites. This observation suggested to Smith that Arber’s hypothesis was correct—the enzyme was cleaving the DNA at specific sequences. In order to prove that this was the case, Smith had to determine the sequence at which the enzyme cleaved the DNA, which he called the recognition site. With the purified enzyme and evidence of sequencespecific DNA cleavage, Smith focused his attention on determining the sequence of the recognition site. At this time, the 1960s, the only known method of DNA sequencing was to sequentially remove nucleotides from the 5 ¿ end of DNA and determine their identity by thin layer chromatography (TLC). Smith devised a scheme to sequence the recognition site by using known enzymes to cleave the ends of a DNA strand into small pieces that could be analyzed by TLC (see Figure). Smith began by labeling the 5 ¿ end of endonuclease Rdigested DNA with a radioactive marker, 32P. This was accomplished by first treating the DNA with alkaline phosphatase, an enzyme that catalyzes the removal of 5 ¿ phosphate groups from polynucleotides. Next, polynucleotide kinase, which catalyzes addition of phosphate to the 5 ¿ end of polynucleotides, was used to transfer 32P from labeled ATP to the terminal nucleotide. Now, the terminal nucleotide could be easily distinguished from the rest of the nucleotides, by virtue of its specific radioactive label. The DNA was then digested to single nucleotides with a nuclease called pancreatic DNase. The only 32P-labeled nucleotides observed contained adenine (A) and guanine (G). Since no 32P-labeled nucleotide containing cytosine (C) Recognition site 3' 5' Endonuclease R 3' 5' P P Alkaline phosphatase 3' 5' Polynucleotide kinase [32P] ATP 3' 5' P* *P Digestion with various nucleases 5' 3' 5' 3' 5' 3' 5' 3' n=1 n=2 n=3 P* P* P* *P *P *P Mononucleotides Dinucleotides Trinucleotides Schematic representation of the method used to determine the nucleotide sequence recognized by endonuclease R. T7 bacteriophage DNA was digested with endonulcease R. After removal of the 5’ phosphate, and addition of a 32P label, the 5’ end-labeled DNA was digested with a variety of nucleases. 32P-labeled mononucleotides, dinucleotides, and trinucleotides were isolated and analyzed to determine the recognition site sequence. [Adapted from T. J. Kelly and H. O. Smith, 1970, J. Mol.Biol. 51:393.] or thymine (T) was detected, Smith deduced that the first base in the recognition sequence must be a purine. To determine the second base in the recognition site, Smith used a nuclease that could not cleave 5 ¿ terminal dinucleotides. In other words, the entire DNA sample was digested into single nucleotides except the final two, which remained in dinucleotide form. Since the DNA previously had been cleaved with endonuclease R, the 5 ¿ terminal dinulceotides are the first two bases in the recognition site. Smith first separated the dinucleotides from the single nucleotides. When he analyzed the dinucleotides by TLC, he found only two species of dinucleotides that carried the 32 P label. The identity of the 32P-labeled dinucleotides was determined by comparing their migration to that of dinucleotides of known sequence. One of the species displayed the same migration as the dinucleotide GA; the other migrated with the dinucleotide AA. Smith concluded that the second base in the recognition sequence was adenine. Analysis of the rest of the recognition site would not be so easy, but Smith’s persistence paid off. He identified the third base in the recognition site as cytosine using a similar, but slightly more complicated method. He further showed this to be the end of the recognition sequence by showing that the fourth nucleotide could contain any base. Now he knew digestion of double stranded DNA with endonuclease R creates several smaller fragments with identical 5’ ends, which contain the sequence purine-adenine-cytosine. Since the DNA strands are complementary, the only possible way this could occur is if the enzyme recognized a six-base sequence that appeared the same on either strand, known as a pallindromic sequence. Therefore, Smith concluded that endonuclease R recognized and cleaved DNA specifically at the sequence GTPyPuAC. Discussion Although the first restriction enzyme had been purified two years before Smith reported his work on endonuclease R, he was the first to demonstrate sequence-specific cleavage. He then went on to purify and characterize the methylase that allows DNA from H. influenzae to escape cleavage. By using these sequence-specific restriction enzymes, researchers could now cleave DNA at specific sites. The impact of restriction enzymes on biological research over cannot be overstated. Early on, these enzymes were used for mapping plasmid and phage DNA. Now they are routinely used for probing the structure of both specific genes and of DNA from individuals. In addition, they are primary reagents in the construction of gene expression vectors, allowing DNA from different sources to be cleaved at specific sequences, then joined with similarly cleaved DNA. The results are seen everyday in laboratories employing recombinant DNA technologies. In 1978, Hamiliton Smith was awarded the Nobel Prize for Physiology and Medicine in recognition of his powerful discovery.