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Home , EcoRI, Endonuclease, Restriction enzyme

NSCASCPPR PERSPECTIVE PAPER: CLASSIC PNAS

How restriction became the workhorses of

Richard J. Roberts* , 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 R showing the 11 dis- zymes unsuitable for use as 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 , 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 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 , 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 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 and could be nicely separated from one an- few known restriction 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 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 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 , 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 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 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 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 . Danna, in Nathans’ acterized by Smith is now called HindII 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. digest on one of Loening’s polyacryl- SV40 DNA, rather more than one amide gels. In those days, most gels would have anticipated by chance. Agarose Gels and More Restriction were prepared in glass tubing, not the Endonucleases slab gels that are common today. Once Mapping DNA One momentous feature of the paper the gel had run, two methods could be Another feature of the Danna and was the realization that gel electro- used to find the resulting DNA, which Nathans paper that helped to make it a phoresis provided a wonderful assay by in this case had been radioactively la- classic was that it clearly foresaw several which one might hope to find new re- beled. The first method was to expel the potential applications of restriction en- striction endonucleases. That this turned gel from the tube and place it in contact donucleases that later proved to be of out to be true is testified by the current with an x-ray film for autoradiography. considerable utility. For instance, the collection of known restriction endo- The result, using 14C-labeled DNA, is authors clearly saw the possibility of us- , which now numbers Ͼ3,600 shown in Fig. 1. The second method was ing these fragments to prepare a physi- individual enzymes representing Ͼ250 to cut the gel into slices and use a scin- cal map of the SV40 genome, a feat that different specificities (18). Of course, tillation counter to quantitate the radio- was later accomplished in Nathans’ lab- electrophoresis in tube gels was soon activity in each slice. The results of this oratory (3). The authors also showed superseded by polyacrylamide slab-gel kind of analysis, in this case, using DNA that it was possible to localize the origin electrophoresis, again first introduced by that had been labeled with 32P, are of replication (4) and to position the Loening. Even more useful were the shown in Fig. 3. One immediate advan- early and late genes of SV40 onto this agarose gels first described in 1972 (19, tage of autoradiography is readily ap- ‘‘restriction map’’ (5), and that any indi- 20) and the use of ethidium bromide to parent. The third and fourth peaks vidual gene could be mapped by testing stain the DNA in them, which permitted shown in Fig. 3 can be seen in the auto- for biological activity during transfor- nonradioactive DNA to be visualized radiograph (Fig. 1) to each contain two mation experiments (15). Even more (21). Initially, these agarose gels also separate fragments labeled CD and EF. insightful was the realization that in- were run in tubes, tapered at the end to The resolution necessary to separate formative mutants could be made by stop the slippery agarose from sliding

5906 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500923102 Roberts Downloaded by guest on September 25, 2021 out, and then run later on vertical slabs, when special frames were made to pre- vent slippage and intricate combs were developed to allow the even loading of samples (22). These gels were, in turn, replaced by the horizontal slab gels we run today (23). Indeed, it was the devel- opment of agarose slab-gel electro- phoresis that enabled my laboratory in the early 1970s to isolate large numbers of restriction endonucleases. At first, we gladly shared these enzymes with the academic community, but the demand quickly became far too high. In 1975, New England Biolabs started to make the sale of these enzymes its major product line, and many other companies soon followed suit. Sequencing DNA Another important use for restriction enzyme fragments was in the early days of DNA sequencing. When Fred Sanger first developed methods for RNA se- Fig. 4. Dan Nathans (left) and Hamilton Smith (right) in the laboratory at Johns Hopkins University quencing, it was because there were (Copyright 1978, Susie Fitzhugh). many small RNA molecules such as tRNAs and 5S RNA on which to prac- tice, which is always crucial when new haps the most prominent development we have now witnessed a multibillion- methods are being developed. However, was the discovery of methods for pre- dollar biotechnology industry develop there were no comparable short DNA paring recombinant DNAs. When John that can trace its origins back to the molecules. This situation changed when Morrow and Paul Berg (26) discovered ideas embedded in the paper submitted restriction fragments became available, that SV40 DNA contained a single site by Danna and Nathans to PNAS in and some of the early primers used by for the restriction endonuclease EcoRI, 1971. DNA diagnostics, DNA forensics, Sanger’s laboratory were the tiny frag- which had been discovered in Herb and the routine checking of DNA sam- ments produced when ␾X174 DNA was Boyer’s laboratory (27), the stage was ples in the research laboratory all arose cleaved by restriction enzymes recogniz- set to convert SV40 DNA into a vector from the methods described in this clas- sic paper. ing four base pairs (24). This methodol- for recombinant DNA. The original method pioneered by Peter Lobban and ogy was underway in the early 1970s, The Nobel Prize and for a time my laboratory was con- Dale Kaiser (28) used bacteriophage ␭ It is particularly fitting that when the tinuously sending restriction enzyme as a vector, but Dave Jackson, Bob Nobel Committee decided in 1978 who samples to the Medical Research Coun- Symons, and Paul Berg (29) used the better method of adding poly(A) tails to should be on the ticket for the discovery cil Laboratory of Molecular Biology of restriction enzymes and their uses, an SV40 vector and poly(T) tails to the (Cambridge, U.K.). The restriction en- they selected Nathans, who joined Arber fragment to be cloned. Herb Boyer and zyme maps also helped in the assembly and Smith (Fig. 4). Arber had provided of DNA sequences by providing useful Stan Cohen soon came up with the even the theoretical framework that described landmarks and permitting manageable better idea for making recombinant the biology of restriction and modifica- segments of DNA to be isolated and DNAs (30). They used DNA to tion and had successfully isolated the sequenced before assembly. The chemi- join a DNA molecule with ‘‘sticky’’ very first type I restriction enzyme, cal sequencing methods developed by ends, produced by cleavage with EcoRI EcoBI. Smith had discovered the first Wally Gilbert and Allan Maxam also restriction endonuclease, to a type II restriction enzyme, endonuclease depended heavily on restriction enzymes DNA molecule that also had been R. But it was Nathans who first realized to provide the unique 5Ј termini, which cleaved by EcoRI, ensuring that the and then demonstrated just how power- could be labeled with 32P before chemi- ends of each DNA molecule were com- ful restriction enzymes could be when cal degradation (25). plementary. This complementarity used for the physical mapping of SV40 provided a very easy route to the prepa- DNA and its genes. This trio laid the Recombinant DNA and Biotechnology ration of recombinant DNA molecules groundwork that has led to the current There are many other developments and enabled any DNA to be cloned into addiction to restriction enzymes as rou- that stem from this early work on re- the easily grown E. coli. From these tine, but essential, tools for molecular striction enzymes and SV40 DNA. Per- early tools of the molecular biologists, biologists.

1. Danna, K. & Nathans, D. (1971) Proc. Natl. Acad. 5. Lai, C. J. & Nathans, D. (1975) Cold Spring Harbor 9. Arber, W. (1965) Annu. Rev. Microbiol. 19, 365– Sci. USA 68, 2913–2917. Symp. Quant. Biol. 39, 53–60. 378. 2. Smith, H. O. & Wilcox, K. W. (1970) J. Mol. Biol. 6. Roberts, R. J. (1976) CRC Crit. Rev. Biochem. 4, 10. Meselson, M. & Yuan, R. (1968) Nature 217, 51, 379–391. 123–164. 1110–1114. 3. Danna, K., Sacks, G. H., Jr., & Nathans, D. (1973) 7. Luria, S. E. & Human, M. L. (1952) J. Bacteriol. 64, 11. Kelly, T. J., Jr., & Smith, H. O. (1970) J. Mol. Biol. J. Mol. Biol. 78, 363–376. 557–569. 51, 393–409. 4. Nathans, D. & Danna, K. J. (1973) Nat. New Biol. 8. Bertani, G. & Weigle, J. J. (1953) J. Bacteriol. 65, 12. Loening, U. E. (1967) Biochem. J. 102, 251– 236, 200–202. 113–121. 257.

Roberts PNAS ͉ April 26, 2005 ͉ vol. 102 ͉ no. 17 ͉ 5907 Downloaded by guest on September 25, 2021 13. Crawford, L. V. & Black, P. H. (1964) Virology 24, 19. Aaij, C. & Borst, P. (1972) Biochim. Biophys. Acta 25. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. 381–387. 269, 192–200. Acad. Sci. USA 74, 560–564. 14. Old, R., Murray, K. & Roizes, G. (1975) J. Mol. 20. Hayward, G. S. & Smith, M. G. (1972) J. Mol. Biol. 26. Morrow, J. & Berg, P. (1972) Proc. Natl. Acad. Sci. Biol. 92, 331–339. 63, 383–388. USA 69, 3365–3369. 15. Lai, C. J. & Nathans, D. (1974) Virology 60, 21. Sharp, P. A., Sugden, B. & Sambrook, J. (1973) 27. Yoshimori, R. N. (1971) Ph.D. thesis (Univ. of 466–475. Biochemistry 12, 3055–3063. California, San Francisco). 16. Lai, C. J. & Nathans, D. (1974) J. Mol. Biol. 89, 22. Sugden, B., DeTroy, B., Roberts, R. J. & 28. Lobban, P. E. & Kaiser, A. D. (1973) J. Mol. Biol. 179–193. Sambrook, J. (1975) Anal. Biochem. 68, 78, 453–471. 17. Jeffreys, A. J., Wilson, V. & Thein, S. L. (1985) 36–46. 29. Jackson, D. A., Symons, R. H. & Berg, P. (1972) Nature 316, 76–79. 23. McDonell, M. W., Simon, M. N. & Studier, F. W. Proc. Natl. Acad. Sci. USA 69, 2904–2909. 18. Roberts, R. J., Vincze, T., Posfai, J. & Macelis, (1977) J. Mol. Biol. 110, 119–146. 30. Cohen, S. N., Chang, A. C. Y., Boyer, H. W. & D. (2005) Nucleic Acids Res. 33, D230– 24. Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, Helling, R. B. (1973) Proc. Natl. Acad. Sci. USA D232. 441–448. 70, 3240–3244.

5908 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500923102 Roberts Downloaded by guest on September 25, 2021