Proc. Natl. Acad. Sci. USAVol. 74, No. 2, pp. 542-546, February 1977Biochemistry

A thermostable sequence-specific endonuclease from Thermusaquaticus

(restriction endonuclease/physical mapping of DNA/DNA nucleotide sequence/bacteriophage OX174/thermophile)SHOWBU SATO*, CLYDE A. HUTCHISON UIPt, AND J. IEUAN HARRISMedical 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, ofnovel specificity has been partially purified from an extremethermophile, Thermus aquaticus. The enzyme cleaves bacter-iophage A DNA at many (>30) sites and bacteriophage OX174RF DNA at 10 sites. The enzyme is active at temperatures uto 700. The cleavage sites on 4X174 RIFDNA have been mrThe sequence recognized and cleaved by Taq I has been s ownto be the symmetrical tetranucleotide:

I,5' T-C-G-A 3'

3' A-G-C-T I'

tEndonucleases that recognize specific base-paired sequencesin DNA have been partially purified from various microorga-nisms (1). Restriction-like enzymes of this general type havebecome indispensable for the physical mapping of genomes andfor DNA sequencing (1, 2), and consequently enzymes withnew specificities are of particular interest. Enzymes from an

extreme thermophile, Thermus aquaticus, are exceptionallystable to heat and to protein-denaturing reagents (3-5), and wehave sought to prepare a thermostable restriction endonucleasefrom this source that would aid in the study of DNA structureand of the mode of action of restriction-like enzymes. We reporthere on the partial purification of a new restriction endonu-clease, Taq I, with a novel specificity, from T. aquaticus (YT-1).The nucleotide sequence recognized by Taq I and the site ofcleavage within that sequence have also been determined.

MATERIALS AND METHODS

Thermus aquaticus (YT-1) cells (6) grown at 70-750 and storedfrozen (at -18°) were obtained from the Microbiological Re-search Establishment, Porton Down, Wiltshire, England. Therestriction enzymes Hae 11 (7), Hae III (8), and Hindll (9) weregifts of N. L. Brown. Escherichia colh DNA polymerase I wasfrom Boehringer Mannheim, West Germany, and T4 DNApolymerase was a gift of R. Kamen. Bacteriophage 4X174 DNAwas derived from the lysis mutant am3 by phenol extractionand the double-stranded form was prepared as described pre-viously (10). 32P-Labeled 4X174 RF DNA was prepared bynick-translation (11) using [a-32P]dATP from New EnglandNuclear Corp. Bacteriophage A was prepared by heat-inductionof CI1s7S7 prophage, and its DNA was obtained by phenol ex-

traction of banded phage (12).

Abbreviation: buffer A, 10mM Tris-HCl/1 mM 2-mercaptoethanol,pH 7.4.* Present address: Mitsubishi Kasei Institute of Life Sciences, 11 Mi-namioya Machida-shi, Tokyo, Japan.

t Present address: Department of Bacteriology and Immunology andCurriculum in Genetics, University of North Carolina, Chapel Hill,N.C. 27514.

Analysis of Restriction Enzyme Fragments. Unlabeled Aor 4X174 RF DNA (1-2 Ag) or 32P-labeled kX174 RF DNA (40ng) was digested with endonuclease at 370 for 2-12 hr in 20 Alof 10 mM Tris-HCl (pH 7.4)/10 mM MgCl2/10 mM 2-mer-captoethanol. Reactions were stopped by adding 10 ll of 100mM EDTA in 30% sucrose containing 0.1% bromphenol blue.The samples were analyzed by electrophoresis on 5% acryl-amide slab gels (14 X 16 X 0.15 cm) in Tnrs-borate/EDTA (13).Gels were stained with ethidium bromide, 0.5 Mg/ml in theTris borate/EDTA buffer, for 30 min and photographed underUV light with a red filter on the lens or covered with Saranwrapand autoradiographed. A similar procedure with 1% agarosegels (7) was used to assay column fractions obtained duringpurification of Tag I. The conditions of DNA digestion and gelelectrophoresis used at different stages are given in legends toFigs. 1 and 2.

Hybridization Mapping of Taq I Fragments of 4X174 RFDNA. Two-dimensional hybridization was performed as de-scribed previously (10). Unlabeled OX174 RF DNA fragmentsproduced by Hae III digestion were hybridized with [32P]-4X174 RF DNA fragments obtained by digestion with TagI.

Identification of Taq I Cleavage Site. The rapid "plus andminus" method of Sanger and Coulson (14) as applied by Brownand Smith (15) was used to identify cleavage sites in [32PIkX174DNA. The details of this procedure are given, with the inter-pretation of the results, in Fig. 6.

Purification of Taq L. T. aquaticus cells (200 g) were thawedin 200 ml of buffer A (10 mM Tris-HCl/I mM 2-mercapto-ethanol, pH 7.4) and disrupted by means of a French pressurecell at 110-140 kg/cm2. The cell extract (3, 4) was diluted withan equal volume of buffer A and centrifuged at 100,000 X gfor at least 60 min to remove as much as possible of a pigmented"slime" that interferes with subsequent purification steps [cf.Sato and Harris (5)]. The clear supernatant fraction was stirredfor 15 min with a suspension of phosphocellulose (50 g ofWhatman P-li in buffer A) which was then collected by fil-tration, washed thrice with 200-ml portions of buffer A, andsubsequently extracted with the same volume of buffer Acontaining 1 M NaCl. The extract (about 600 ml) was broughtto 40% saturation with solid ammonium sulfate and centrifuged;the supernatant phase was brought to 90% saturation withammonium sulfate. The resulting precipitate was resuspendedin buffer A, dialyzed extensively against several changes of thesame buffer, centrifuged to remove insoluble protein, and ap-plied (in a volume of about 50 ml) to a column (20 X 1.8 cm)of phosphocellulose equilibrated with buffer A. The columnwas developed with a linear gradient (800 ml) to 0.8 M NaCl.Effluent fractions (5 ml) were tested for endonuclease activityby- the gel electrophoresis method (cf. ref. 7), and active frac-tions, eluting between 0.35 and 0.45 M NaCl (55-67, Fig. 1,left) were pooled and concentrated to about 3 ml by pressure

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Proc. Natl. Acad. Sci. USA 74 (1977) 543

: 3 *- 51 5 59 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) fromthe 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. Thisconcentrate was subjected to gel filtration on a Sephadex G-200column (45 X 1.5 cm) in buffer A. Effluent fractions (2 ml) werecollected and maximum endonuclease activity was located infractions 24-26 (Fig. 1, right). Ovalbumin (molecular weightabout 48,000) elutes in approximately the same position, whichsuggests that Taq I may have a native molecular weight of about40,000-50,000.

RESULTSPurification of Taq I. The active fractions appeared to be

free of contaminating nonspecific nucleases and gave sharp andcharacteristic gel electrophoresis patterns with both A DNA andOX174 DNA, even after prolonged periods (up to 16 hr) of di-gestion. The fractions eluted from phosphocellulose tended togive more diffuse gel patterns (Fig. 1, left), but after preheatingto 700 for 10-15 min (a step that appeared to cause preferentialinactivation of contaminating nucleases) these fractions alsogave sharp gel patterns with OX174 DNA, similar to those ob-tained subsequently (Fig. 1, right) with the Sephadex G-200fractions.The yield of Taq I (about 4000 units from 200 g of frozen

cells, a unit being defined as the amount required to give thelimit gel pattern in 1 hr at 37° with 1 gg of DNA) appears low

1 2 3_T,1 3w

-2 Ti

T2 _- Z 1 --ZtZ2

~ -7Z Z3

Z 2

5; ~~~~~~~~~~~~Z 3

.T4~ ~ ~ TZ7

lo _ TZ6 Z65 ^*_ TZ 7 __z7

* *,* Z8- Z8TZ9

T76 TZ10

TZ11 --Z9

.T8

- T 9-T1O

- TZi2

- -TZ13 --Zi18 - -_-Z1Z4

FIG. 2. Gel electrophoresis of products of cleavage of OX174RF DNA by Taq I. Left, 2 jg of OX174 RFDNA digested to comple-tion with Taq I. Right, [32P]JX174 RF DNA (40 ng) completely di-gested by Taq I (channel 1), Taq I plus Hae III (channel 2), or HaeIII (channel 3).

by comparison with yields of other restriction-like endonuc-leases from bacterial sources (e.g., ref. 7). In this procedure,however, an appreciable proportion of endonuclease activitywas discarded in the fraction of the cell extract that did notadsorb to phosphocellulose., (This fraction does absorb toDEAE-cellulose.) It should also be noted that the estimated yieldis based on digestions carried out at a temperature (370) thatis some 350 below the optimal growth temperature of T.aquaticus (cf. refs. 3 and 4).

Taq I Fragments of 4X174 RF DNA. Unlabeled DNA wascompletely digested with Taq I and the digest was fractionatedby electrophoresis on a 5% acrylamide gel. Ten prominentbands, designated T1 to T1O from the high-molecular-weightend of the gel, were observed (Fig. 2, left). To determine the

Table 1. OX174 RF DNA restriction fragments

Taq I Hae III Double-digestion-fragments fragments* fragments

T1 2820Zi 1300 TZ1 (=Z1)

T2 1250Z2 1050

TZ2 930 Z2:T1Z3 870Z4 600 TZ3 (= Z4)

T3 420T4 330

Z5 320Z6a 290 TZ5a (= Z6a)Z6b 285 TZ5b (=Z6b)

TZ6 270 Z3:T4TZ7 250 Z5:T3

T5 230 Z7 230Z8 190

TZ8 170 Z8:T3TZ9 145 Z7:T5

T6 140 TZ10 (=T6)Z9 115 TZ11 (=Z9)

T7 90 TZ12 (=T7)Z10 73 TZ13 (= Z10)

T8 60 TZ14t 60T9 36T10 25

Produced by digestion with Taq I, with Hae III, and with a mixtureof both; fragment lengths expressed in nucleotide pairs were estimatedfrom electrophoretic mobility on a 5% acrylamide gel (13).* Length of Hae III fragments was from (16).t Multiplet.

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Hae III No

Zl Z2 Z3 Z4 Z5 Z6 Z7 Z8

i,.~~~~~ Hi 1211~~~~~~~~~~

- _ _ _ _ *_ 1 my T3H p ---a- be T4

s_ _ _ ~T6T7

-1 - - ~ - - - 1T8

FIG. 3. Two-dimensional hybridization mapping of Taq I frag-ments of 4X174 RF DNA. The diagram on the right is an interpre-tation of the fingerprint.

sizes of the Taq I fragments, digests of cX174 RF DNA withother restriction endonucleases, Hae II, Hae III, and HindII,were fractionated in the same manner. A standard curve re-lating fragment size to electrophoretic mobility could then beconstructed, using known values for the marker fragment sizes(16). The size of fragments T2 through T10 was estimated fromthis standard curve and the size of T1 was calculated by sub-tracting the sum of T2 to T10 from the total length, 5400 nu-cleotide pairs, of the 4X174 RF DNA. The sizes of the Taq Ifragments in nucleotide pairs are listed in Table 1.Cleavage of 4X174 RFDNA by Taq I plus Hae III. Dou-

ble-digestion with a mixture of Taq I and Hae III was carriedout using 32P-labeled DNA (Fig. 2, right). Hae III fragmentsZ2, Z3, Z5, Z7, and Z8 (8) appear to be cut by Taq I; Taq Ifragments T1, T2, T3, T4, and T5 were cut by Hae III. Dou-ble-digestion products TZ2, TZ4, TZ6, TZ7, TZ8, TZ9, andTZ14 were produced by the combined action of both enzymes;the intensity of band TZ14 suggests that it might contain morethan one fragment. Fragments Z1, Z4, Z6a, Z6b, Z9, Z10, T6,T7, and T8 remained unchanged. The order of the Hae IIIfragments (16) is Z3-Z7-Z5-Z4-Zl-Z2-Z6b-Z6a-Z9-Z1O, in whichthe Hae III fragments not cleaved by Taq I are in italics. Be-cause the segment Z4-Zi, which contains no Taq I site, is largerthan T2, it must be within fragment T1. Similarly, the segment

a b c d e

=-7 314

-

NT6

1 ~~~~~~~~~~7T8

FIG. 4. Partial digestion of 4X174 RF DNA (3 ,g) with Taq I at370. Channels: a, 5 min; b, 10 min; c, 30 min; d, 60 min; e, 300 min.

Z6b-Z6a-Z9Z10, which is larger than T3, must be included infragment T2.Two-Dimensional Hybridization Mapping. In order to

identify the Hae III fragments that shared nucleotide sequenceswith Tag I fragments, Hae III fragments of unlabeled OX174DNA and Taq I fragments of 32P-labeled OX DNA were hy-bridized two-dimensionally (10) (Fig. 3). The partial order ofthe Taq I fragments of OX174 DNA could then be establishedto be T1-T6-T2-(T7,T8)-T4-T5-T3 (Table 2). Fragments T7and T8 could not be ordered in this way because both are con-tained within Z3. Hybridization products involving fragmentsT9 and T10 were not observed on the gels, presumably becauseof their small size.

Partial Digestion of 4X174 RF DNA with Taq I. To locatefragments TV and TIO, kX174 RF DNA was partially digestedwith Taq I. The resulting products were fractionated on a 5%acrylamide gel (Fig. 4) and their sizes were estimated from asemilogarithmic plot of the number of nucleotide pairs againstthe electrophoretic mobility of the limit Taq I products T1 toT10 (Table 3). The limit product composition of some partial

Table 3. 4X174 RF DNA fragments produced by partialdigestion with Taq I

Products Length Possible composition of partial(nucleotide products (sum of length of

Limit Partial pairs) constituents)

Table 2. Deduced order of Taq I fragments fromtwo-dimensional hybridization to Hae III fragments (10)

Taq I fragmentsoverlapped

Tl

Z2 T1, T6

Z3 T2,T4,T7,T8

Z4 Ti

Z5 T3, T5

Z6 (a,b) T2

Z7 T4, T5

Z8 T3

Rearranged in map order

Hae III Taq I

Zi

Z2

Z6

Z3

Z7

Z5

Z8

Z4

T3

T4T5

T6

I 460420

T4 + T8 + T9 + T10 (451)

J 400 T4 + T9 + T10 (391),T4 + T8 (390)

K 355 T4 + T10 (355),T5 + T7 + T9 (356)

330230

L 200 T7 + T8 +T9 +TIO (2T6 + T9 + T10 (201)

M 180 T7 + T8 + T9 (186),T6 + T9 (176)

N 145 T7 + T8 (150),T7 + T9 + T10 (151)

1400 110

T7T8T9T10

90603625

211),

T7 + T10 (115),T8 + T9 + T10 (111)

Hae III

OrCZ

Hae IIIfragment

Zi

Taq I fragment order deduced: T1-T6-T2-(T7,T8)-T4-T5-T3.

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1000 2000 3000 4000 5000

T8T9 T10T6

J5T4,T5, T3 T1 l, T2 ,T7TZ81

TZ6, Z79 TZ2 TZ4

TZ14

Z3 Z7Z5 Z8 Z4 Zi Z2 Z6b .Z6a Z9 Z10

FIG. 5. Map of the Taq I fragments of OX174 RF DNA. The TaqI fragments are aligned with the Hae III map. The ends of the mapare drawn at the single Pst I cleavage site (15).

products could be established from their sizes when consideredin the light of the known partial order of Taq I fragments. Apossible composition for product L, T6 + T9 + T1O, was ex-cluded by the known composition of product 0 (Table 3); thissuggested that T10 was located close to T7 or T8, both of whichhybridize with Hae III fragment Z3. T6, on the other hand, islocated within Hae III fragment Z2, known to be far distantfrom Z3 in the DNA sequence. It therefore may be concludedthat fragment L comprises T7, T8, T9, and T1O, which are alllocated in a cluster between T4 and T2 within Hae III fragmentZ3. The only possible compositions of the partial products K,J, and I indicate that T1O occurs adjacent to T4 and that thesequence in fragment L is T7-T8-T9-T1O. The order of the 10Taq I fragments on the 4X174 DNA is thereby uniquely es-tablished as T1-T6-T2-T7-T8-T9-T1O-T4-T5-T3.Alignment of the Taq I and Hae III Maps. In order to lo-

cate the exact map positions of the Taq I fragments relative tothe known positions of Hae III fragments, the double-digestionproducts were reexamined. Fragments T5 and Z7, which areof similar size and hybridize to each other, would be expectedto give rise to two nonoverlapping parts of equal size. These arenow likely to occur in the "multiplet" band TZ14 which cor-responds to about 60 nucleotide pairs. A map of Taq I frag-ments, consistent with the results of the Taq I/Hae III double

a

0. Cz -A-G-C -T +A+G+C +T 0z Oz

digestion and constructed by fixing the T4/T5 cleavage site ata locus within 27 and 60 np distant from Z3, is shown in Fig. 5.An alternative alignment obtained by placing the T4/T5 site60 nucleotide pairs within Z3 can be ruled out because it would,for example, predict that Taq I would not cleave within frag-ment Z5.The Recognition Sequence and Cleavage Site of Taq I. A

provisional nucleotide sequence for OX174 DNA has been de-termined in this laboratory (F. Sanger et al., unpublished data).From this we were able to predict the sequence recognized byTaq I simply by inspection of the nucleotide sequences in theparts where Taq I sites had been mapped. It was thereby notedthat the sequence T-C-G-A occurred exclusively at the 10locations indicated by the mapping experiments. The exactlocation of the T-C-G-A sequences within the DNA sequencewas used to calculate the sizes of Taq I fragments and theseproved to be in good agreement with the values obtained by thegel electrophoresis procedure (Table 1).

In order to identify the exact site of cleavage within therecognition sequence (as well as to obtain independent con-firmation that Taq I recognizes the sequence T-C-G-A), weused the method of Brown and Smith (15). One such experi-ment is illustrated in Fig. 6, which shows that cleavage with TaqI produces ends with the structure

-ToH 3'

-A-G-Cp 5'

Hence, the cleavage site is

T-TG-A- 3'

-A-G-C-T- 5'

1

QT TCz -A -G -C -T

A

C

TCAA

CC----

+A+G+C +TP7OzFIG. 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 of these were subjected to the plus-and-minus system (14) with Hae III digestion. Controlsamples (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 HaeIII. All samples were analyzed by electrophoresis on a 12% acrylamide/7 M urea slab gel and autoradiographed. The figure shows the relevantportion 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 of the gel, although bands are missing in the -T and +C positions. Sample TOZ gives a strongband 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 thenewly synthesized (complementary) strand and demonstrates that the Taq I enzyme has cleaved after the T residue. The main band in sampleTCz 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 thecomplementary strand after cleavage at the Taq I site. Since the extension continues only to the cleaved end of the opposite (viral) strand, theband identifies the cleavage site in the viral strand. The results are explained as follows:

TOZ

32P-product-TOHtemplate-A-G-Cp

TCZ

T4 polymerase T-C.GOH 3'

-A-G-p 5'

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DISCUSSIONThe restriction endonuclease Taq I recognizes and cleaves ata new symmetrical tetranucleotide sequence and is a usefuladdition to the group of restriction enzymes now available forstudy of DNA structure. In this connection it has already proved-valuable in the determination of the complete sequence ofbacteriophage 4X174 DNA (F. Sanger et al., unpublisheddata).

In common with other enzymes from T. aquatcs previouslystudied in this laboratory (3-5), Taq I is stable and active attemperatures of up to 700. Contaminating nonspecific exo- andendonucleases are inactivated at this temperature and theavailability of a thermostable restriction enzyme greatly extendsthe range of conditions that may be used to study DNA struc-ture and the mode of action of restriction endonucleases.The availability of DNA of known nucleotide sequence has

greatly facilitated the identification of restriction -enzymerecognition sites. Thus it has become possible to deduce therecognition sequences of enzymes that cleave 4X174 RF DNAsimply by correlating a map of restriction fragments with theknown nucleotide sequences. The specificities for Hae II (B.G. Barrell and P. M. Slocombe, personal communication), HinfI(C. A. Hutchison, III and B. G. Barrell, unpublished data), Pst1 (15), and now Taq I, as reported here, were all predicted inthis way prior to verification by direct experiment.We thank Dr. F. Sanger and colleagues for providing unpublished

nucleotide sequences of 0X174 DNA. We also thank Dr. N. L. Brownfor gifts of restriction enzymes and advice on enzyme assay, and Dr.R. Kamen for a gift of T4 polymerase. We thank A. Atkinson for in-forming us that he independently discovered a restriction-like activity

toward X DNA in extracts of T. aquaticus YT-1. S.S. was a visitingscientist from the Mitsubishi Kasei Institute, Tokyo, Japan, and C.A.H.was supported by U.S. Public Health Service Career DevelopmentAward AI-70604.

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