Proc. Natl. Acad. Sci. USA Vol. 74, No. 2, pp. 542-546, February 1977 Biochemistry A thermostable sequence-specific endonuclease from Thermus aquaticus (restriction endonuclease/physical mapping of DNA/DNA nucleotide sequence/bacteriophage OX174/thermophile) SHOWBU SATO*, CLYDE A. HUTCHISON UIPt, AND J. IEUAN HARRIS Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England Communicated by F. Sanger, November 29,1976

ABSTRACT A sequence-specific endonuclease, Taq I, of Analysis of Restriction Enzyme Fragments. Unlabeled A novel specificity has been partially purified from an extreme or 4X174 RF DNA (1-2 Ag) or 32P-labeled kX174 RF DNA (40 thermophile, Thermus aquaticus. The enzyme cleaves bacter- at 370 for 2-12 hr in 20 iophage A DNA at many (>30) sites and bacteriophage OX174 ng) was digested with endonuclease Al RF DNA at 10 sites. The enzyme is active at temperatures u of 10 mM Tris-HCl (pH 7.4)/10 mM MgCl2/10 mM 2-mer- to 700. The cleavage sites on 4X174 RIF DNA have been mr captoethanol. Reactions were stopped by adding 10 ll of 100 The sequence recognized and cleaved by Taq I has been s own mM EDTA in 30% sucrose containing 0.1% bromphenol blue. to be the symmetrical tetranucleotide: The samples were analyzed by electrophoresis on 5% acryl- amide slab gels (14 X 16 X 0.15 cm) in Tnrs-borate/EDTA (13). I, Gels were stained with ethidium bromide, 0.5 Mg/ml in the 5' T-C-G-A 3' Tris borate/EDTA buffer, for 30 min and photographed under UV light with a red filter on the lens or covered with Saranwrap 3' A-G-C-T I' and autoradiographed. A similar procedure with 1% agarose t gels (7) was used to assay column fractions obtained during purification of Tag I. The conditions of DNA digestion and gel Endonucleases that recognize specific base-paired sequences electrophoresis used at different stages are given in legends to in DNA have been partially purified from various microorga- Figs. 1 and 2. nisms (1). Restriction-like enzymes of this general type have Hybridization Mapping of Taq I Fragments of 4X174 RF become indispensable for the physical mapping of genomes and DNA. Two-dimensional hybridization was performed as de- for DNA sequencing (1, 2), and consequently enzymes with scribed previously (10). Unlabeled OX174 RF DNA fragments new specificities are of particular interest. Enzymes from an produced by Hae III digestion were hybridized with [32P]- extreme thermophile, Thermus aquaticus, are exceptionally 4X174 RF DNA fragments obtained by digestion with Tag stable to heat and to protein-denaturing reagents (3-5), and we I. have sought to prepare a thermostable restriction endonuclease Identification of Taq I Cleavage Site. The rapid "plus and from this source that would aid in the study of DNA structure minus" method of Sanger and Coulson (14) as applied by Brown and of the mode of action of restriction-like enzymes. We report and Smith (15) was used to identify cleavage sites in [32PIkX174 here on the partial purification of a new restriction endonu- DNA. The details of this procedure are given, with the inter- clease, Taq I, with a novel specificity, from T. aquaticus (YT-1). pretation of the results, in Fig. 6. The nucleotide sequence recognized by Taq I and the site of Purification of Taq L. T. aquaticus cells (200 g) were thawed cleavage within that sequence have also been determined. in 200 ml of buffer A (10 mM Tris-HCl/I mM 2-mercapto- ethanol, pH 7.4) and disrupted by means of a French pressure MATERIALS AND METHODS cell at 110-140 kg/cm2. The cell extract (3, 4) was diluted with Thermus aquaticus (YT-1) cells (6) grown at 70-750 and stored an equal volume of buffer A and centrifuged at 100,000 X g frozen (at -18°) were obtained from the Microbiological Re- for at least 60 min to remove as much as possible of a pigmented search Establishment, Porton Down, Wiltshire, England. The "slime" that interferes with subsequent purification steps [cf. restriction enzymes Hae 11 (7), Hae III (8), and Hindll (9) were Sato and Harris (5)]. The clear supernatant fraction was stirred gifts of N. L. Brown. Escherichia colh DNA polymerase I was for 15 min with a suspension of phosphocellulose (50 g of from Boehringer Mannheim, West Germany, and T4 DNA Whatman P-li in buffer A) which was then collected by fil- polymerase was a gift of R. Kamen. Bacteriophage 4X174 DNA tration, washed thrice with 200-ml portions of buffer A, and was derived from the lysis mutant am3 by phenol extraction subsequently extracted with the same volume of buffer A and the double-stranded form was prepared as described pre- containing 1 M NaCl. The extract (about 600 ml) was brought viously (10). 32P-Labeled 4X174 RF DNA was prepared by to 40% saturation with solid ammonium sulfate and centrifuged; nick-translation (11) using [a-32P]dATP from New England the supernatant phase was brought to 90% saturation with Nuclear Corp. Bacteriophage A was prepared by heat-induction ammonium sulfate. The resulting precipitate was resuspended of CI1s7S7 prophage, and its DNA was obtained by phenol ex- in buffer A, dialyzed extensively against several changes of the traction of banded phage (12). same buffer, centrifuged to remove insoluble protein, and ap- plied (in a volume of about 50 ml) to a column (20 X 1.8 cm) Abbreviation: buffer A, 10 mM Tris-HCl/1 mM 2-mercaptoethanol, of phosphocellulose equilibrated with buffer A. The column pH 7.4. was developed with a linear gradient (800 ml) to 0.8 M NaCl. * Present address: Mitsubishi Kasei Institute of Life Sciences, 11 Mi- Effluent fractions (5 ml) were tested for endonuclease activity namioya Machida-shi, Tokyo, Japan. t Present address: Department of Bacteriology and Immunology and by-the gel electrophoresis method (cf. ref. 7), and active frac- Curriculum in Genetics, University of North Carolina, Chapel Hill, tions, eluting between 0.35 and 0.45 M NaCl (55-67, Fig. 1, N.C. 27514. left) were pooled and concentrated to about 3 ml by pressure 542 Downloaded by guest on September 26, 2021 Biochemistry: Sato et al. Proc. Natl. Acad. Sci. USA 74 (1977) 543

: 3 *- 51 5 5 9 63 67 71 s VI I I

Pnos5;Floce-i,5 Sephadex G-200 FIG. 1. Assay of endonuclease activity (cf. ref. 7). Samples (2 gl) from the phosphocellulose column fractions (5 ml) or samples (1 gl) from the G-200 column fractions (2 ml) incubated with 2 jg of A DNA for 2 hr at 37°. Numbers above refer to fraction numbers.

dialysis in an Amicon cell with a UM-10 membrane. This by comparison with yields of other restriction-like endonuc- concentrate was subjected to gel filtration on a Sephadex G-200 leases from bacterial sources (e.g., ref. 7). In this procedure, column (45 X 1.5 cm) in buffer A. Effluent fractions (2 ml) were however, an appreciable proportion of endonuclease activity collected and maximum endonuclease activity was located in was discarded in the fraction of the cell extract that did not fractions 24-26 (Fig. 1, right). Ovalbumin (molecular weight adsorb to phosphocellulose., (This fraction does absorb to about 48,000) elutes in approximately the same position, which DEAE-cellulose.) It should also be noted that the estimated yield suggests that Taq I may have a native molecular weight of about is based on digestions carried out at a temperature (370) that 40,000-50,000. is some 350 below the optimal growth temperature of T. aquaticus (cf. refs. 3 and 4). RESULTS Taq I Fragments of 4X174 RF DNA. Unlabeled DNA was I. The active to be completely digested with Taq I and the digest was fractionated Purification of Taq fractions appeared Ten free of contaminating nonspecific nucleases and gave sharp and by electrophoresis on a 5% acrylamide gel. prominent bands, designated T1 to T1O from the high-molecular-weight characteristic gel electrophoresis patterns with both A DNA and end of the gel, were observed (Fig. 2, left). To determine the OX174 DNA, even after prolonged periods (up to 16 hr) of di- gestion. The fractions eluted from phosphocellulose tended to give more diffuse gel patterns (Fig. 1, left), but after preheating Table 1. OX174 RF DNA restriction fragments to 700 for 10-15 min (a step that appeared to cause preferential inactivation of contaminating nucleases) these fractions also Taq I Hae III Double-digestion- gave sharp gel patterns with OX174 DNA, similar to those ob- fragments fragments* fragments tained subsequently (Fig. 1, right) with the Sephadex G-200 fractions. T1 2820 Zi 1300 TZ1 (=Z1) I 4000 units 200 g of frozen The yield of Taq (about from T2 1250 cells, a unit being defined as the amount required to give the Z2 1050 limit gel pattern in 1 hr at 37° with 1 gg of DNA) appears low TZ2 930 Z2:T1 Z3 870 1 2 3 Z4 600 TZ3 (= Z4) _T,1 3w T3 420 T4 330 -2 Ti Z5 320 T2 _- Z 1 --Zt Z6a 290 TZ5a (= Z6a) Z2 Z6b 285 TZ5b (=Z6b) ~ -7Z Z3 TZ6 270 Z3:T4 5 3 TZ7 250 Z5:T3 Z 2 T5 230 Z7 230 .T4 ~ ~ ~ TZ7 Z8 190 lo _ TZ6 Z6 5 ^*_ TZ 7 __z7 TZ8 170 Z8:T3 * *,* Z8- Z8 TZ9 145 Z7:T5 TZ9 6 T6 140 TZ10 (=T6) T7 TZ10 TZ11 --Z9 Z9 115 TZ11 (=Z9)

- TZi2 90 TZ12 .T8 T7 (=T7) - - TZ13 --Zi1 Z10 73 TZ13 (= Z10) - T 9 8 - -_-Z1Z4 T8 60 TZ14t 60 -T1O T9 36 T10 25 FIG. 2. Gel electrophoresis of products of cleavage of OX174 Produced by digestion with Taq I, with Hae III, and with a mixture RF DNA by Taq I. Left, 2 jg of;OX174 RF DNA digested to comple- of both; fragment lengths expressed in nucleotide pairs were estimated tion with Taq I. Right, [32P]JX174 RF DNA (40 ng) completely di- from electrophoretic mobility on a 5% acrylamide gel (13). ~~~~~~~~~~~~Z * gested by Taq I (channel 1), Taq I plus Hae III (channel 2), or Hae Length of Hae III fragments was from (16). III (channel 3). t Multiplet. Downloaded by guest on September 26, 2021 544 Biochemistry: Sato et at. Proc. Nati. Acad. Sci. USA 74 (1977)

Hae III Hae III No a b c d e Zl Z2 Z3 Z4 Z5 Z6 Z7 Z8

11~~~~~~~~~~ i,.~~~~~ Hi 12 -= 7 3 14

Or CZ - _ __ _ *_ 1 my T3 - H p ---a- be T4 NT6 1 s_ _ _ ~T6 T7 T8 -1 - - ~ - - - 1T8 ~~~~~~~~~~7 FIG. 4. Partial digestion of 4X174 RF DNA (3 ,g) with Taq I at 60 FIG. 3. Two-dimensional hybridization mapping of Taq I frag- 370. Channels: a, 5 min; b, 10 min; c, 30 min; d, min; e, 300 min. ments of 4X174 RF DNA. The diagram on the right is an interpre- tation of the fingerprint. Z6b-Z6a-Z9Z10, which is larger than T3, must be included in fragment T2. sizes of the Taq I fragments, digests of cX174 RF DNA with Two-Dimensional Hybridization Mapping. In order to other restriction endonucleases, Hae II, Hae III, and HindII, identify the Hae III fragments that shared nucleotide sequences were fractionated in the same manner. A standard curve re- with Tag I fragments, Hae III fragments of unlabeled OX174 lating fragment size to electrophoretic mobility could then be DNA and Taq I fragments of 32P-labeled OX DNA were hy- constructed, using known values for the marker fragment sizes bridized two-dimensionally (10) (Fig. 3). The partial order of (16). The size of fragments T2 through T10 was estimated from the Taq I fragments of OX174 DNA could then be established this standard curve and the size of T1 was calculated by sub- to be T1-T6-T2-(T7,T8)-T4-T5-T3 (Table 2). Fragments T7 tracting the sum of T2 to T10 from the total length, 5400 nu- and T8 could not be ordered in this way because both are con- cleotide pairs, of the 4X174 RF DNA. The sizes of the Taq I tained within Z3. Hybridization products involving fragments fragments in nucleotide pairs are listed in Table 1. T9 and T10 were not observed on the gels, presumably because Cleavage of 4X174 RF DNA by Taq I plus Hae III. Dou- of their small size. ble-digestion with a mixture of Taq I and Hae III was carried Partial Digestion of 4X174 RF DNA with Taq I. To locate out using 32P-labeled DNA (Fig. 2, right). Hae III fragments fragments TV and TIO, kX174 RF DNA was partially digested Z2, Z3, Z5, Z7, and Z8 (8) appear to be cut by Taq I; Taq I with Taq I. The resulting products were fractionated on a 5% fragments T1, T2, T3, T4, and T5 were cut by Hae III. Dou- acrylamide gel (Fig. 4) and their sizes were estimated from a ble-digestion products TZ2, TZ4, TZ6, TZ7, TZ8, TZ9, and semilogarithmic plot of the number of nucleotide pairs against TZ14 were produced by the combined action of both enzymes; the electrophoretic mobility of the limit Taq I products T1 to the intensity of band TZ14 suggests that it might contain more T10 (Table 3). The limit product composition of some partial than one fragment. Fragments Z1, Z4, Z6a, Z6b, Z9, Z10, T6, T7, and T8 remained unchanged. The order of the Hae III Table 3. 4X174 RF DNA fragments produced by partial fragments (16) is Z3-Z7-Z5-Z4-Zl-Z2-Z6b-Z6a-Z9-Z1O, in which digestion with Taq I the Hae III fragments not cleaved by Taq I are in italics. Be- Products Length Possible composition of partial cause the segment Z4-Zi, which contains no Taq I site, is larger (nucleotide products (sum of length of than T2, it must be within fragment T1. Similarly, the segment Limit Partial pairs) constituents)

Table 2. Deduced order of Taq I fragments from I 460 T4 + T8 + T9 + T10 (451) two-dimensional hybridization to Hae III fragments (10) T3 420 J 400 T4 + T9 + T10 (391), Rearranged in map order T4 + T8 (390) Hae III Taq I fragments K 355 T4 + T10 (355), fragment overlapped Hae III Taq I T5 + T7 + T9 (356) T4 330 Zi Tl Zi T5 230 Z2 T1, T6 Z2 L 200 T7 + T8 +T9 +TIO (2211), T6 + T9 + T10 (201) Z3 T2,T4,T7,T8 Z6 M 180 T7 + T8 + T9 (186), T6 + T9 (176) Z4 Ti Z3 N 145 T7 + T8 (150), T7 + T9 + T10 (151) Z5 T3, T5 Z7 T6 140 Z6 (a,b) T2 Z5 0 110 T7 + T10 (115), T8 + T9 + T10 (111) Z7 T4, T5 Z8 T7 90 T8 60 Z8 T3 Z4 T9 36 T10 25 Taq I fragment order deduced: T1-T6-T2-(T7,T8)-T4-T5-T3. Downloaded by guest on September 26, 2021 Biochemistry: Sato et al. Proc. Nati. Acad. Sci. USA 74 (1977) 545 1000 2000 3000 4000 5000 digestion and constructed by fixing the T4/T5 cleavage site at T8T9 T10 T6 a locus within 27 and 60 np distant from Z3, is shown in Fig. 5. J5T4,T5, T3 T1 l, T2 ,T7 An alternative alignment obtained by placing the T4/T5 site TZ81 60 nucleotide pairs within Z3 can be ruled out because it would, TZ6, Z79 TZ2 TZ4 for example, predict that Taq I would not cleave within frag- TZ14 ment Z5. Z3 Z7Z5 Z8 Z4 Zi Z2 Z6b . The Recognition Sequence and Cleavage Site of Taq I. A Z6a Z9 Z10 provisional nucleotide sequence for OX174 DNA has been de- FIG. 5. Map ofthe Taq I fragments of OX174 RF DNA. The Taq termined in this laboratory (F. Sanger et al., unpublished data). I fragments are aligned with the Hae III map. The ends of the map From this we were able to predict the sequence recognized by are drawn at the single Pst I cleavage site (15). Taq I simply by inspection of the nucleotide sequences in the parts where Taq I sites had been mapped. It was thereby noted that the sequence T-C-G-A occurred exclusively at the 10 products could be established from their sizes when considered locations indicated by the mapping experiments. The exact in the light of the known partial order of Taq I fragments. A location of the T-C-G-A sequences within the DNA sequence possible composition for product L, T6 + T9 + T1O, was ex- was used to calculate the sizes of Taq I fragments and these cluded by the known composition of product 0 (Table 3); this proved to be in good agreement with the values obtained by the suggested that T10 was located close to T7 or T8, both of which gel electrophoresis procedure (Table 1). hybridize with Hae III fragment Z3. T6, on the other hand, is In order to identify the exact site of cleavage within the located within Hae III fragment Z2, known to be far distant recognition sequence (as well as to obtain independent con- from Z3 in the DNA sequence. It therefore may be concluded firmation that Taq I recognizes the sequence T-C-G-A), we that fragment L comprises T7, T8, T9, and T1O, which are all used the method of Brown and Smith (15). One such experi- located in a cluster between T4 and T2 within Hae III fragment ment is illustrated in Fig. 6, which shows that cleavage with Taq Z3. The only possible compositions of the partial products K, I produces ends with the structure J, and I indicate that T1O occurs adjacent to T4 and that the sequence in fragment L is T7-T8-T9-T1O. The order of the 10 3' Taq I fragments on the 4X174 DNA is thereby uniquely es- -ToH tablished as T1-T6-T2-T7-T8-T9-T1O-T4-T5-T3. -A-G-Cp 5' Alignment of the Taq I and Hae III Maps. In order to lo- cate the exact map positions of the Taq I fragments relative to Hence, the cleavage site is the known positions of Hae III fragments, the double-digestion products were reexamined. Fragments T5 and Z7, which are of similar size and hybridize to each other, would be expected to give rise to two nonoverlapping parts of equal size. These are T-TG-A- 3' now likely to occur in the "multiplet" band TZ14 which cor- -A-G-C-T- 5' responds to about 60 nucleotide pairs. A map of Taq I frag- ments, consistent with the results of the Taq I/Hae III double 1

A T T 4w*44P 40- C lo. I a AC A .-OP 410 4m 4m C-- C --

0. Cz -A-G-C -T +A+G+C +T Oz +A+G+C +TP 0z QT TCz -A -G -C -T 7Oz FIG. 6. Cleavage site sequences for Taq I. Hae III fragment Z6b was used as a primer on OX174 viral strand as template, according to the standard conditions for the "plus and minus" method (14). After the unincorporated triphosphates were removed by gel filtration, the synthesized [32P]DNA fraction was divided into 12 portions. Eight ofthese were subjected to the plus-and-minus system (14) with Hae III digestion. Control samples (OT and Oz) were simply incubated with the enzymes Taq I and Hae III, respectively. Sample TOZ was incubated with both enzymes. Sample TCZ was incubated with four deoxyribonucleoside triphosphates, T4 DNA polymerase, and the tvwo restriction enzymes, Taq I and Hae III. All samples were analyzed by electrophoresis on a 12% acrylamide/7 M urea slab gel and autoradiographed. The figure shows the relevant portion of the autoradiograph with a diagram showing the distribution of the bands and the sequence deduced from the plus-and-minus results. The sequence T-C-G-A can be read off in the center ofthe gel, although bands are missing in the -T and +C positions. Sample TOZ gives a strong band in the same position as the band in the +T sample. This is the fragment extending from the Hae III cleavage site to the Taq I site on the newly synthesized (complementary) strand and demonstrates that the Taq I enzyme has cleaved after the T residue. The main band in sample TCz is in the same position as the bands in the -A and +G samples. This product is formed by extension, by the T4 DNA polymerase, of the complementary strand after cleavage at the Taq I site. Since the extension continues only to the cleaved end of the opposite (viral) strand, the band identifies the cleavage site in the viral strand. The results are explained as follows: TOZ TCZ

32P-product-TOH T4 polymerase T-C.GOH 3' template-A-G-Cp -A-G-p 5' Downloaded by guest on September 26, 2021 546 Biochemistry: Sato et al. Proc. Natl. Acad. Sci. USA 74 (1977) DISCUSSION toward X DNA in extracts of T. aquaticus YT-1. S.S. was a visiting scientist from the Mitsubishi Kasei Institute, Tokyo, Japan, and C.A.H. The restriction endonuclease Taq I recognizes and cleaves at was supported by U.S. Public Health Service Career Development a new symmetrical tetranucleotide sequence and is a useful Award AI-70604. addition to the group of restriction enzymes now available for study of DNA structure. In this connection it has already proved 1. Nathans, D. & Smith, H. 0. (1975) Annu. Rev. Blochem. 44, -valuable in the determination of the complete sequence of 273-293. bacteriophage 4X174 DNA (F. Sanger et al., unpublished 2. Murray, K. & Old, R. W. (1974) in Progress in Nucleic Acid Research and Molecular Biology, ed. Cohn, W. E. (Academic data). Press, New York), pp. 117-185. In common with other enzymes from T. aquatcs previously 3. Hocking, J. D. & Harris, J. I. (1973) FEBS Lett. 34, 280-293. studied in this laboratory (3-5), Taq I is stable and active at 4. Hocking, J. D. & Harris, J. I. (1976) in Enzymes and Proteins temperatures of up to 700. Contaminating nonspecific exo- and from Thermophilic Microorganisms, ed. Zuber, H. (Birk- endonucleases are inactivated at this temperature and the hiuser-Verlag, Basel), Experientla Supplement, Vol. 26, pp. availability of a thermostable restriction enzyme greatly extends 121-135. the range of conditions that may be used to study DNA struc- 5. Sato, S. & Harris, J. I. (1976) Eur. J. Biochem., in press. ture and the mode of action of restriction endonucleases. 6. Brock, T. D. & Freeze, H. (1969) J. Bacteriol. 98,289-297. The availability of DNA of known nucleotide sequence has 7. Roberts, R. J., Breitmeyer, J. B., Tabachnik, N. F. & Meyer, P. A. (1975) J. Mol. Biol. 91,121-123. greatly facilitated the identification of restriction -enzyme 8. Middleton, J. H., Edgell, M. H. & Hutchison, C. A., III (1972) J. recognition sites. Thus it has become possible to deduce the Virol. 10, 42-50. recognition sequences of enzymes that cleave 4X174 RF DNA 9. Smith, H. 0. & Wilcox, K. W. (1970) J. Mol. Biol. 51, 379- simply by correlating a map of restriction fragments with the 391. known nucleotide sequences. The specificities for Hae II (B. 10. Hutchison, C. A., III (1977) NucleIc Acids Res., in press. G. Barrell and P. M. Slocombe, personal communication), HinfI 11. Maniatis, T., Jeffrey, A. & Kleid, D. G. (1975) Proc. Natl. Acad. (C. A. Hutchison, III and B. G. Barrell, unpublished data), Pst Sci. USA 72, 1184-1188. 1 (15), and now Taq I, as reported here, were all predicted in 12. Reznikoff, W. S., Winter, R. B. & Hurley, C. K. (1974) Proc. NatI. this way prior to verification by direct experiment. Acad. Sci. USA 71, 2314-2318. 13. Peacock, A. C. & Dingman, C. W. (1968) Biochemistry 7, We thank Dr. F. Sanger and colleagues for providing unpublished 668-674. nucleotide sequences of 0X174 DNA. We also thank Dr. N. L. Brown 14. Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448. for gifts of restriction enzymes and advice on enzyme assay, and Dr. 15. Brown, N. L. & Smith, M. (1976) FEBS Lett. 65,284-287. R. Kamen for a gift of T4 polymerase. We thank A. Atkinson for in- 16. Jeppesen, P. G. N., Sanders, L. & Slocombe, P. M. (1976) Nucleic forming us that he independently discovered a restriction-like activity Acids Res. 3, 1323-1340. Downloaded by guest on September 26, 2021