SiteSpecificDeoxyribonuclease Produced by a Marine Bacterium,

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Agrie.

2107 --2109, 19g8 BioL Chet}!.,52 (8).

2107

Note

Then, the dialyzcd.specimenwas equi]ibrated

of

'I'heenzyme cencentraLed. "`lls'pl named 1604", The

I 16-04

was

Hiroshi CHiuRA, Yukihiko NoRo, KANAyAMA,' Y6ko UEDA,' Shiriji

about

7.000

unitsig

definedas the amount digest 1ug of' Adcm

Usio SIMIDu"' and Jun TAKAGI

put

on

an

1･'buffer

heparin-agarose(Bio-Rad. linear Nt KCI gradient of O

U.S.A,) and eluted with u inF buli'er, The activily was eluted at O.45 M KCI eoncentration. pcuk

SiteSpecific Deoxyribonuclease Produced by a Marine Bacterium, )Etavobacteriuin

co]umn

-･O,7

a singlc symmetrical

as

itwas dialyzedund

and

thus

spccimcn

obtained

yie!dof this purified

ce]]s, when of enzyme

DNA

onc

unil

sufTieienl

at

was

spccimcn

of uctivity

is

te cumpletely

30"C in 60min in

the

mixture containing SOmM {Sigma Ltd.).10ms･iMgCl,. 40mM KCI, and 5mM 2-mercaptoKagaku Co,. Ltd.),The enzymatic acDeparrm"nt qf'Bietog.1', Dii'isifm ttf'' NalrwaiS('ienc'es,ethano] (Nakarai tivitlrwas exainined by the Agarose g.elelectropheresjs Iniernationai ChristianLiniveJ'sitl', method using a Tris-boratebutTersystem.S} The cleavage ,Viraka, 7'ok.i't) 181,Japan "･as unu]yzed by Gene Co., Lld., Ton.va-mctchi, puttern el' 1d('n7DNA -'ith thc cnzyme that the fragment sizcs coina cemputer, and suggested To,}'ama 93(1, Japaii **Lahoratory this suggestion, Oc'ean Re,t'uai'ch fnstiture, eided to thttt of EeoRII. To confirm (V' i64, (PX174RF! DNA was digested with the enzyme. and 7he C,'niversii.v 7bk,vo, Nakano-ktv, 71)kyo Japan qf' 2･.7]7hp and 2-,619bp fragments were which produccd 15. 1988 ReceiN･edFcbruar}' supported the suggestion that the enzyme Es'pl1604 isan isoschizomeret' EcoRII, The clet"･age site ot' Flspl ]604 was idcntified b>, the Escherichia coii JMIOS and dideoxy method9) using Iiindll, more than ]]･O kinds of Sincethe discoverv ot' ' of the eleetroencoliphage )vll3,Based on an examination site-specific endodeoxyribonuc]eases {restrietion the that Eypl16C)4recognizes zymes)i'3' have been iso]atedfrom somc 600 spceies ol' phorctogrum, -e concluded as EcoRII.tO'iii but as same bauteriaund reported. A]I of thern have been obtained pentunucleotide sequence indicutedbelow, itinduces cleavage at a differentposition from terrestrial bacteria cxeept Fokl isolatedfrorn a te that ot' BstN than EcoRII [ICCCA,iT)GG] buli(lentical marinebacterium,FVavohad'teriumokeaiiokoiies,4)Furtherhave not been as extensiK,ely 1 [CCI(A.i'T)GGI,i!) a site-specificendodeoxyribonuclettse more. the marine bacteria studied as the terrestria] bactcria.Under these circumstances. we have been attcmpting to surve}, site-specific of the laborator}, endonucleascs in the marine bagtcria TABLE I, PROPERTIES OI, 1'H[, STTE-SPEC/IHC Institute,University collection of the Oceun Research ENDoNucLF.AsE fispI1604 PRoDucED of Tokyo. Two out of 68 preparations,i.e,,those from BY P, MARINE BACTER]UM I 16-04 rvaT,ohacterium F7avohacteriuinI 16-04 and I 16-11.showed apprcciable restriction endonuc]ease-likc activitics,S"") XVe tried to Optimum the enzymc fretn filt"'()htwteJ'iumI 16-04. Thc purif'y C 20-40 Temp., was ineculatcd into u modified sea water microorganisrn 8.S pHMgi eontaining O. 1 Proteosc No, 3. bt'oth (PI)ES-tl) pcptonc 5-4020 conc,, mM Bacte lr･east extracl, and O.t'',, O.2"･"Pelypcpton, O.1'l,ieldot' the '. conc,, mM: Mg" and optimum ce]]s was 3,5gi'1, The eells -･ere suspended in the same u.os･--o.lo Ca] buffer, disrupLed by honication. and centrit'uged al O.Ot --O.IO Co!i 30.000rpm for 1hr. The supernatun[ thLs obtained was --1.o e.o.h-. Mn2+ dia]yzedagainst F buiTer{10mber KPOa, 1 mM 2-mercapo,o]-v1.o Zn2+ 'I'he toethanol. pH 7.S,5.e"'･i, specimen g]},ccrin}, diafy'zed was of' PhosphocelluloscPL 1 (Whatman, put on a column Retalned activitv at U.K.)which was equilibrated with F bufferinadvance and 3o 1.ooomM Na:, "i, -eluted with a lineargrudientof O O.8M KCI in F buffer. 52 Molecular Mass. K daltons Relatis,e active material, as cxamined en JLdcvn' CC't(A,iT)GG An enzymatically Cleavagc sitc at DNA. -･as recovcrcd (CS-methylated-cytosinc-frec) * ismethylated. cleavuge actis,e When indicutedC" position around O.45M KCI. Thc fracrionsef enzymatically is unable to occur. material were combined and dial},zedagainst }" buffer.

Tris-HCI

reaction

Co..

','Vippon

,Vi('i'ohiologl',

'1'.

,

'

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2108

H. CHiuRA et ",+

FiG, 1, Cleavage

of

aL

12345678910111213i415

Zdcm' DNA

with

Diderent Sodium Concentration.

The reaction was done using 4units ot'cachenzyme for S hr at 30 C. pH 8,5forEs'pl 1604and 60'C. pH 7.4for BstNI in the assay mixture containing ] gtgof substrate 2 dc'niJDNA, SOmM Tris- HCI (SigmaCo,, Ltd,), 5mM 2-mcrcaptoe[hanol (NakaraiKagaku Co,, Ltd.).suitable umounts of MgC]2 with differentconcentrations ofNaCl, and ended with a stop rnixture (1OO mM EDTA, 60e･i, sucrose. 2`t{1 s.odium dodecy] sulfate and 2?,:bromophenolblue,pH 7.5},then electrophoresed on a O.7?i, (SDS), agarose gel using the TBE buffer system at constant current, 12,5mA. for 15hr. Ethidium bromide was addcd bcfore the start of the eleetrophoresis to a finalconcentratien ofO,75 sigf'ml,Detailsof the sodium concentration are summarized in "F" and the followingtable. are Fspl 1604 and BstNI. "O" represents the optjmum condition for each "bk""means only the substrate was enzyme, and te the luncsindicateden the put on. Numbcrs correspond "B"'

plcture. Satnple conditions Run ili L 2.4,

ENZ.

bkF,

Mg,

mM

Na

Mg.

Na.

rnM

7.57.5,7.5,7.5,7,5,7,5,7.5,7.5.

O

F 6. F 8.10.12.14.F FFF

mM

o

75 15e

5.

B 7. B 9,IL13,15. BBB

300 6001200

]o,]o,IO,IO,10,10.10. 150 o 41 75 300 6001,200

B

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2109

SitcSpccificDNase from Marine BacteriumNavohaclerium srearoth(,rinophilus, Although,DNA produced by Ba('iUu,s scission with BstNI isnot afl'ected by the methylation of the targct scquence, when Espl 1604 is considcred, the DNA cleavage islikely to be inhibited by meth}'lation of

the

cytosine

residue

the target

at

sequence.

Thisisbased

(1970). Acids Res.,12, r167 (19g4). 3) R. J,Roberts,Nuctcei`r 4) H.Sugisaki, Gene,3, 17C1978). 5) H. Chiura, J. Takagi and U. Simidu,Abstracts of Papcrs, thc Annual Meeting of the Agricu[tura[

discretefragmcnts were not frorn ;. dam-(,N"-mcthylated-adeninc-free)DNA produced as well as diim' (,Nb-methylated-adenine), In contrast, ;.dcm' DNA gave idcnticalsharp discrctefragmcnts on a electropherogram for either EeoRII or BstNI, In experiments conducted to this date, purifiedEspI 1604 has

Chemical Society of Japan, Sapporo, July, t985, p, 356.6) I･{.Chiura. J. Takagi, T. Naito and U. Sirnidu, Abstractsof Papers,the JointAnnual Meeting of the Japanese Society of Genetics and the Japanese Society of Molecular Biology. Nagoya, Dcccmbcr. shown negligible exonucleolytic activity in any 1986, p. 297. practical sense. The enzymatic or Ev)I 1604 are sum7) H, Chiura, J. Takagi, Y. Noro. S. Kanayama, Y. properties marized in Table I,The tnarine bacterium Flavoha{'tef'ium Ueda and U, Sirnidu,Abstracts of Papers, the I 16-04 produces significant amounts of a site-spceific AnnualMeet{ngoftheAgriculturalChemicalSociet}' endodeoxyribonuclease, Es?)I1604,which iseasily purificd of Japan, Tokyo, April,19g7,p. I12. and charactcrizcd b>'the followingproperties/The Mg2' Manual g) R. W. Davi$, D. Botstein and J. R. Roth, for Engineering. Advunced Buctcria] ionrequirement forthe endonuclease actix,ttyisreplaceGenetic Genetics':Cold Spring Harbor Laboratory. Ncw abie with several divalenr cations, and the enzyme shows a York, 1980, pp. 148- 152. high tolerance for salt concentration (seeFig. I),These Vol. 101, ArpI 1604 wM provide a usefu1 9) J. Messing, in Enzymolog},,'' propertiesof the enzyme -tool for gene manipulation even under circumstances Academic Press,New York, 1983,pp. 20 78. which would be critical for other enzvmes, 10) R. N. Yoshimori, Ph.D. thesis,1971. 11) C. H. Bigger.K. Murray and N. E, Murray. Nature New Biology,244, 7 (1973). REFERENCES 12) M, Ehrlich,M, A, Gama-Sosa, L. H. Carreria,L. G, Ljungdhal. K. C. Kuo and G. W. Gehrke. Gene, 13, 1) H, O. Smith and K. W. Wilcox, J. MoL Biol.,51, 379 1399 (1985). on

the

observation

that

clear

`'A

LLMethods

(l970).

2) T. J. Kelly and

H. O. Smith, J. MoL

BioL, 51, ]93

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