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How Restriction Enzymes Became the Workhorses of Molecular Biology

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How Restriction Enzymes Became the Workhorses of Molecular Biology PNAS CLASSIC PAPER: PERSPECTIVE How restriction enzymes became the workhorses of molecular biology Richard J. Roberts* New England Biolabs, 32 Tozer Road, Beverly, MA 01915 Restriction enzymes have proved to be invaluable for the physical mapping of DNA. They offer unparalleled opportunities for diag- nosing DNA sequence content and are used in fields as disparate as criminal forensics and basic research. In fact, without restriction enzymes, the biotechnology industry would certainly not have flourished as it has. The first experiments demonstrating the utility of restriction enzymes were carried out by Danna and Nathans and reported in 1971. This pioneering study set the stage for the mod- ern practice of molecular biology in which restriction enzymes are ubiquitous tools, although they are often taken for granted. oday, it is difficult to imagine a acterized are now known as the type I time when our laboratory freez- systems. Although these enzymes recog- ers were not well stocked with nize specific DNA sequences, they have restriction enzymes, when DNA the unfortunate property of cleaving Tsequencing was not possible, or when Fig. 1. Radioautogram of 14C-labeled SV40 DNA DNA randomly, thus rendering the en- genes were only accessible to the geneti- cleaved with endonuclease R showing the 11 dis- zymes unsuitable for use as cloning and cists and could not be simply cloned out tinct fragments (figure 3 from ref. 1; courtesy of the mapping reagents. Nathans family and Kathleen Danna). by recombinant DNA technology. Yet, A significant breakthrough came in in December 1971, a key paper ap- 1970 when the first of two papers from peared in PNAS that set the stage for mapping of the origin of replication (4) Smith’s laboratory described an enzyme, much of what is now routine (1). In that and the location of SV40 genes (5). endonuclease R, that was able to cleave paper, Kathleen Danna and Daniel These pioneering studies set the stage bacteriophage T7 DNA into specific Nathans of Johns Hopkins University for modern molecular biology. Suddenly, fragments (2). This was the first type II (Baltimore) showed for the first time everyone wanted to map DNA and use restriction enzyme, the sort that now that the restriction enzyme called ‘‘en- any available restriction enzymes to ex- populates our freezers, because it recog- donuclease R,’’ discovered by Hamilton amine their favorite genome (in those nize specific sequences and also gives Smith and Kent Wilcox (2), could be days, usually a phage or viral DNA). rise to very specific cleavage. Smith had used to produce specific fragments of When I moved from Harvard to Cold been looking for an enzyme that might simian virus 40 (SV40) DNA. Moreover, Spring Harbor Laboratory (Cold Spring be involved in site-specific recombina- the authors showed that these fragments Harbor, NY) in 1972, I first purified the tion in Haemophilus influenzae and could be nicely separated from one an- few known restriction endonucleases and thought at first that endonuclease R other by electrophoresis on a polyacryl- then began looking for more, ideally might be his long-sought quarry. With amide gel. The resulting picture (Fig. 1) ones that would recognize different se- Tom Kelly, he went on to determine the provided an immediate visual example quences and so permit the many appli- DNA sequence recognized by endonu- of just how powerful the combination of clease R and reported it as GTY2RAC restriction endonucleases and gel elec- cations that we routinely see today. Because of the ubiquitous distribution of (11). This sequence seemed too short trophoresis would be. Earlier that year, I for a recombination enzyme, and during had been fortunate to listen to a semi- these enzymes, we were successful be- yond our wildest dreams (6). correspondence with his close friend nar given by Nathans at Harvard Medi- Nathans, who ran the neighboring labo- cal School (Boston) and immediately Restriction Enzymes ratory but was away on sabbatical, it began to think of the possibilities. It was became clear that this enzyme might a defining moment in my life when I The phenomenon of restriction and modification was first observed geneti- have very practical uses for the analysis realized that my half-formed plans for of DNA. future research would be dropped and a cally in 1952–1953 by Luria and Human (7) and Bertani and Weigle (8), al- new avenue pursued. It was because of Polyacrylamide Gel Electrophoresis this presentation that I developed my though they referred to it as host- Nathans realized that the sucrose gradi- own lifelong passion for restriction induced, or host-controlled, variation. ents, which Smith had used to analyze endonucleases. The authors observed that several dif- the reaction products (Fig. 2), might not Looking back at the Danna and ferent bacteriophages varied in their be the best way to try to characterize Nathans paper today, one is struck by ability to grow on different host strains. the specific fragments of DNA produced the simplicity and elegance of the exper- However, once growth was achieved on by cleavage with endonuclease R. The iments. As with all great pioneering one strain, the phages could continue to gradients simply lacked the resolution work, one can say, ‘‘But how obvious!’’ grow happily on this strain but were that would enable the fragments to be Yet, at the time, Smith, who had discov- now restricted in their ability to grow on ered and characterized endonuclease R, other strains. It was not until the 1960s separated, characterized, and used for had not immediately recognized the that a theory to explain this phenome- non was proposed and then biochemi- value of an enzyme that could cleave This Perspective is published as part of a series highlighting DNA specifically. It was Nathans who cally demonstrated by Werner Arber landmark papers published in PNAS. Read more about made the key intuitive leap and then and his laboratory (summarized in ref. this classic PNAS article online at www.pnas.org͞ went on to demonstrate not only that 9). Simultaneously, Matt Meselson and classics.shtml. the resulting fragments could be used to Bob Yuan also isolated a restriction en- Abbreviation: SV40, simian virus 40. produce a physical map of SV40 (3), but zyme from Escherichia coli K (10). The *E-mail: [email protected]. also that this physical map allowed the systems that Arber and Meselson char- © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500923102 PNAS ͉ April 26, 2005 ͉ vol. 102 ͉ no. 17 ͉ 5905–5908 Downloaded by guest on September 25, 2021 them is simply not available by slicing gels. Danna and Nathans proceeded to work out very good length estimates for each of these fragments based on the percentage of the total SV40 DNA that was present and by using the known molecular mass of SV40 DNA of Ϸ3 ϫ 106 kDa, which corresponds to Ϸ5,200 base pairs (13). The team very carefully compared sedimentation values, radioac- tivity measurements, and even EM length measurements to make sure that the results from each method were consistent. 32 Because the recognition sequence of Fig. 3. Electrophoretic analysis of P-labeled endonuclease R was GTYRAC, Danna SV40 DNA cleaved with endonuclease R. After elec- trophoresis, individual slices from the gel were and Nathans expected cleavage to take quantitated by scintillation counting (figure 2 from place approximately once every 1,000 ref. 1; courtesy of the Nathans family and Kathleen base pairs. It was a little surprising, Danna). therefore, that the 4,500 base pairs of SV40 DNA would be cut into 11 frag- ments. Danna and Nathans considered deleting one or more of the specific the possibility that the SV40 DNA was fragments (16). These deletions and heterogeneous. However, the careful others were easily visualized by restric- length measurements of the fragments tion enzyme analysis because fragments precluded this possibility. We now know were either missing completely or ren- that it was, in fact, the original prepara- dered shorter if the deletion was located tion of endonuclease R that was hetero- within them. This feature quickly be- 3 geneous, because it contained not one came a standard use of restriction Fig. 2. Sucrose gradient analysis of H-labeled enzyme maps. In a sentence that fore- SV40 DNA cleaved with endonuclease R (figure 1 restriction enzyme, but two. This discov- from ref. 1; courtesy of the Nathans family and ery was made in several laboratories, shadows the current diagnostic use of Kathleen Danna). including Smith’s, as soon as DNAs restriction endonuclease digestion, the other than T7 DNA were used as assay authors noted: ‘‘By this means, we have substrates. It turns out that, by chance, found that the DNA of small-plaque, mapping. Nathans thus turned to an- bacteriophage T7 DNA has recognition large-plaque, and minute-plaque SV40 other technique, polyacrylamide gel sites for only one of the two restriction strains show specific differences in the electrophoresis, whose use had been pi- enzymes present in the endonuclease R mobility of particular DNA fragments.’’ oneered by Ulrich Loening (12) to sepa- preparation. The original enzyme char- The length variations, now known as rate RNA species. Danna, in Nathans’ acterized by Smith is now called HindII restriction fragment length polymor- laboratory, quickly performed a simple (recognition sequence GTY2RAC), phisms (RFLPs), found in human mini- experiment. She took a small amount of whereas the second enzyme, for which satellites and used so successfully by the purified SV40 DNA being studied in there are no sites in T7 DNA, is HindIII. Alec Jeffreys for forensic purposes (17), his laboratory, incubated it with some Its recognition sequence is A2AGCTT are one of the applications known even endonuclease R, and ran the resulting (14), and this enzyme has six sites in to the general public.
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