.I. .IYd. Hid. (I!l’ii)

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I in

I,ETTER

Two Sequence-specific

TO

THE

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Endonucleases

from Moraxella

bovis

Two new sequence-specific endodeoxyribonucleases have been partially purified from Moralella bovis. These restriction-like enzymes, Mb01 and MboII, each cleave bacteriophage lambda DNA and adenovinls-2 DNA at more than 50 sites. MhoI recognizes the sequcncca

a~ld cleaves at the sites indicated by the arrows. A specific endonuclease, Mosl. has also been purified from lClorarella osloenis aII(I rr,coynizes the same seqnencr as Mb0 1. Several sequence-specific endonucleases havci hcen described which recognize tetranucleotide sequences (Roberts, 1976 and references thrrein). They cleave most DKAs frequently and have proved invaluable in generating small fragments of DNA suitable for sequence analysis. The discovery and subsequent purification of neu specific endonucleases has been greatly aided by an a.ssay utilizing agarose slab gel rlectrophoresis (Aaij & Borst, 1972: Sharp et aZ.. 1973: Sugden et al., 1975). We now endonucleases. report the isolation and characterization of nr\;r restriction-like Mbol and MbolI from Moraxella boeis and Mosl from Xorazella osloen.sis. M. bovis. ATCC 10900, was grown at 37°C on brain/heart infusion (Difco) supplrmented with 2 pg of nicotinamide adenine dinucleotide per ml and 10 pg of hemin per ml. Cultures were harvested in stationary phase and the cells (20 g wet weight from 4 1) were disrupted by sonication at 0°C in 50 ml of 0.01 M-Tris. HCl (pH 7*5), 0.01 NI2-mercaptoethanol. After centrifugation (100,000 g for 2 h), the supernatant wax treated with freshly prepared 571” streptomycin sulfate (20 ml) and stirring was cont inued for 30 minutes. Following centrifugation (10.000 g for 40 min), the supernatant was adjusted to 50% saturation by the gradual addition of solid ammonium sulfate and stirring was continued overnight following the procedure described by Taylor (1953). After centrifugation (10.000 g for 60 min). the precipitate (50P fraction) was dissovlved in 5 ml of 1.0 ivr-NaCl, 0.01 M-Tris. HCl (pH 7.9), 0.01 x-2-mercaptoethanol (Biogel buffer) and dialyzed against Biogel tJuffer for at least two hours. The supernatant fraction from the ammonium sulfate precipitation was adjusted to 700,, satura.tion by the addition of solid ammonium sulfate and the precipitated pro&in (70P fraction) was collected and treated as described above for the 50P fraction. The 5OP fraction was enriched in MboI and the 7OP fraction in MboII. It was found most satisfactory to process each fraction separat,ely through the first phosphocellulose column step. since this facilitated both the assays and the separation of thus large amounts of non-specific nucleases. Both fractions wercl loaded onto srparatcs columns (50 cm s 2.5 cm diam.) of BioGel A-O.5 m (BioRad). previously equilibrated with Biogel buffer. Elution was with Biogel buffer and samples (5 ~1) of column f’raetions (5 ml) were assayed for endonuclease artivitv by a short incubation (30 min) lvith sdenovirlrs-2(Ad-2) DNA or lambda DNA (2 pa). All fractions containing

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EDITOR

171

endonucleolytic activity were pooled (separately for each column) and dialyzed exhaustively against a buffer containing 0.01 M-potassium phosphate (pH 74). 0.01 M-2-mercaptoethanol, 0.001 bf-Na,EDTA, 1006 glycerol (PC buffer). The 50P and 70P fractions were loaded onto separate columns (15 cm x 2 cm diam.) of phosphocellulose (Whatman Pll) p reviously equilibrated with PC buffer and then washed with PC buffer (50 ml). Upon elution with PC buffer containing 0.3 M-KC1 (75 ml) Mb01 eluted immediately, closely followed by an exonuclease as shown in the inset to Figure 1. The MboI activity is best assayed on a substrate such as Bd-2 DNA which is free of modified bases, since lambda DKS, which is partiall! methylated when grown in Escherichia coli K12, is completely cleaved (see also Marinus & Morris. 1975). During the early stages of the purification of MboI. it is essenbial that the extent of digest’ion by MboI is limited so as not to obscure the separation of MboI and the exonuclease. Elution of these columns was continued with 200 ml (total volume) of a linear gradient from 0.3 M to 1.0 M-KC1 in PC buffer and the assay of this column is shown in Figure 1. Mb011 elutes between 0.58 w-KC1 and 0.64 M-KCl. Occasionally Mb011 may be used directly off the phosphocellulose column if it is sufficiently concentrated. More often, fractions containing Mb011 from both the 50P and 70P phosphocellulose columns are pooled. dialyzed against’ PC buffer and loaded onto a column (25 cm x 1.2 cm diam.) of DEAE-cellulose (Whatman DE52) prrviously equilibrated with PC buffer. The column is washed with PC buffer (50 ml) and then eluted with 200 ml (total volume) of a linear gradient from 0 to 0.3 M-KC1 in PC buffer. The assay of fractions from this column is shown in Figure 2. M6oII elutes between 0.05 M and 0.12 M-KCl. Peak fractions of MhoII (36 to 41) were judged to be essentially free of contaminating non-specific nucleases, because DKA samples digested with a tenfold excess of the enzyme for long periods gave sharp bands. with no smearing, on agarose gels. These fractions were combined, concentrated by dialysis against PC buffer containing 5076 glycerol and stored at -20°C. Fractions from both the 50P and 70P phosphocellulose columns containing MhoI were pooled, dialyzed against PC buffer and loaded onto a column (25 cm x 1.2 cm diam.) of phosphocellulose previously equilibrated with PC buffer. The column was washed with PC buffer (50 ml) and then eluted with 200 ml (total volume) of a linear gradient from 0 to 0.35 ~-Kc1 in PC buffer. The assay of this column is shown in Figure 3(a). The Mb01 recovered from this column was routinely purified further by chromatography on a third column (25 cm x 1.2 cm diam.) of phosphocellulose as described above but eluted with 200 ml (total volume) of a linear gradient from 0 to 0.3 M-KCl. The assay of this column is shown in Figure 3(b). Peak fractions of

FIG. 1. Assay of MboI and Mb011 activities from the phosphocellulose column. Portions (5 ~1) of column fractions (6 ml) were incubated at 37°C for 4.5 h in a reaction mixture (60 ~1) containing 2 pg lambda DNA, 6 rniu Tris . HCl (pH 7.9), 6 mM-Mg&, 6 mM-2.mercaptoethanol. Digestion was stopped by the addit,ion of 5 ~1 of a solution containing 5(/o sodium dodecyl sulfate, 0.1 M-xia2EDTA: 15 ~1 of a solution containing 50 9’0 sucrose and 0.2%) bromophenol blur were added and the samples were loaded onto a 1.47” agarose slab gel 20 cm x 20 cm .j 0.4 cm (Sugden of crl.. 1975). Thr gel was subjected to electrophoresis at 150 V for 2.5 h in the presencc~ of 0.5 rg ethidium bromide/ml and photographed under ultraviolet light wit,h Polaroid film (Type 55 P/N). Fraction numbers are given above each channel in the gel. Th P 0.3 M-KU elution was started with fraction 41, and the gradient was started with fraction 73. In total, 118 fractions were collrctrd. Inset: portions (2 ~1) of the indicated fractions were assayed as described above with 2 pg .-k-L’ DNA for 5 min at 3’7°C and fractionated as described above.

1, ETTEK

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i:l

JfboI (55 t,o 59) were judged t,o be essentially free of contaminating non-specific nuclrases. hecause Ad-2 DS;1 samples digested with a tenfold excess of the enzyme> for long periods gave sharp bands. wit’h no smearing. on agarose gels. These fract’ions ww combined. concentrated 1)~. dialysis against I’(’ buffer containing 50° o pl?;crrol and stored at -20°C. The overall recovery of Mhol and Mb011 could not be quantitated with respect to the amount present after sonication because of the presence of contaminating non-specific nucleases. From 20 g of cells the yields of Mb01 and MboII were about 4500 units and 2800 units, respect,ively. One unit of XboI or Mb011 would completely digest’ 1 pg of Ad-2 DNA in one hour at 37°C or 4 pg in four hours. Neither enzyme showed any loss of activity after storage for six months at -20°C. M. o&~msis, ATCC 19976, was grown as described above for M. bovis and the specific endonuclease MosI was isolated using a procedure similar to that previously described for the isolation of rlZu1 from drthrobacter Zutous (Roberts et al., 1976a). In brief, the cells were disrupted by sonication and the liberated proteins fractionated by gel tiltration on BioGel. Peak fracbions of MosI were further purified by chromatography on phosphocellulose. eluting 13ith a gradient from 0 to 1.0 M-KCl. MosI eluted between 0.31 M and 0.35 M-KCl. Peak fractions were then applied to a column (25 cm x 1.2 cm diam.) of aminopentyl Sepharose (Shaltiel & Er-El, 1973) equilibrated in PC buffer. Elution was with 200 ml (total volume) of a linear gradient from 0 to I .O M-KU. MosI eluted between 0.18 M and 0.20 IX-KCl. At t’his stage, Mod was still contaminated with non-specific nuclease activit,y but was sufficiently pure to demonstrate by mixed digestion t’hat MosI and MboI recognized the same sequence (Fig. 4. slots 7 to 15). Mbol and Mb011 were charact,erized by their action on bacteriophage lambda DNA. adenovirus-2 DNA, and simian virus 40 (SV40) DSA (Fig. 4). Both enzymes cleave these DNAs frequently, suggesting that they recognize short sequences. In the case of Mhol, double digests with BumHI and BgZII (Fig. 4, slot 16-25) could not be distinguished from digests with MboI alone. It thus appeared that the sites of cleavage by R~wzHI and those BgZIt were a subset of the sites cleaved by MboI. BanlHl recognizes the sequence G-G-A-T-C-C (Roberts et al.. 1976b) and BgZII recognizes the sequence A-G-A-T-C-T (Pirrotta, 1976) and it seemed likely that the central te0ranucleot’ide G-A-T-C was the recognition sequence for Mbol. Direct evidence for thr recognition sequence and the site of cleavage was obtained using methods previousI> described in detail (Roberts et al., 1976a,b). In brief, adenovirus-2 DNA (5 pg) was digested with MboI and dephosphorylated with alkaline phosphatase (an essential step). The 5’-termini were labeled with bacteriophage T4 polynucleotide kinase (RichardTon, 1965) and [Y-~~P-]ATP (1500 C’/1 mmol : Glynn & Chappell, 1964). Labeled FIG. 2. Assay dicated fractions lrgrnd to Fig. 1. were rollected in

of Mb011 activity were assayed by The gradient wai all.

from the DEAE-cellulose column. incubat,ion with 2 pg lambda DNA started while fraction 1K was being

Portions (5 ~1) of the illfor 4 h as described in thti collected, and 70 fractions

FIG. 3. (a) Assa,v of MboI activity from the second I)hosl)hocellulo~r column. Portions (2 ~1) of the mdicatrd fractions were assayrct by incubation with 1 pg Ad-:! I>XA for 30 min as described 111 the legend to Fig. 1. The gradient \vai started while fraction 30 wa; brsing collected, and 83 fraction. wew taken in all. (b) Assay of Mbol activity from the third phosphoct~llrdose column. Portions (5 ~1) of the. mdloated fractions were assayed by incubation with 2 pg Ad-2 I)NA for 4 h as described in thr were taken in all. Irgrn(l to Fig. 1. The gradirmt was started with fraction 27. and 6ti fractions

I,E’I“I’ER

TO

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I~:l)I’I’oK

1s;

dlhol fragments were then digested for two hours at 37 C with 1 pg of pancreatic DSaw in 5 ~1 of 10 miw-MgCl,, 5 mM-2-mercaptort’hanol. 10 mx]-Tris. HCl (pH 79) and thr products fingerprinted (Brownlee & Sanger. 1969). The results are shown in I:iguw 5, where pancreatic DNase fingerprints of 5’-32P-lal~elcd Mhol fragments art vomparrd with the corresponding fingerprints of 5’- “21’-lahclcd Hnrn.H 1 fragments. It ih immrdiat~ely apparent that the 5’-terminal tctranucleotide G-T-A-C is common to Iwt h wt’s of fragments. In the case of J~hol 1 the specificity is lost at bhe fifth position. sinw all four possible pentanucleotidcs p-(:-A-T-C-S arc found. Each of thr clti,~onrlc,l~otidCR present in the n//ho1 fingerprint (I$. 5) was further analyzed k)~. t rt~atrnrwt~ \vith venom phosphodiesterase to idcntil. t hc 5’.terminal nucleotide and I)y tlipc&on with exonuclease I to ident,ify the 5’-twminal dinucleotide. The results ()t’ t h(hh(s t~xpwimonts were identical with thaw previously reported in t’hc analysis of t trc, 5’.tcwninal oligonucleotides found after digwtion with &w/H 1 (Roberts ct ~1.. I!KW~) and confirmed the 5’-terminal dinuclwtidv as l&A. Prom thcw rrwlts. WV ~~~~~~~twlv that JfhoI rccognizrs the t~,tranucl~,otid(~ palindromc~

\\ it tr t 11thsitw of cleavage indicat)ed 1)~ ttlca arrov.~. .110.91 must also recognize thib lmt w: do not kno\v if t,ht site of’ clrwvag:c~ \\ ithin t ht. recognition sequrnw i?- at t hv same point as that found for ~Z~hoI. sinw our preparatjions of il;losI wcw i~lsufSc*it~ntly pure for us to analyze Mosl fra,gmrnts t)y this method. Because Xbol &3’ll(‘l’iltt‘S fragments hearing a 5’-terminal tt,t,l,ibnnclcotitlr extension G-AT-(‘. itl(wt iwt \\ it,11 that formed by BnmHl and Rrybi 1. vwtors designed for BawHl I’rapnrt~nts may also hc used t,o clorw J~lhol fragmc~nts. .\ttvmpts t,o characterize the syucnce recogniwtl hy :Wholl using a similar approach Aowd no specificity at the 5’.trhrminal nucleotidc and WY \vcrc thus unable to get i\ tly informat~ion about the sequence recognized t)y this enzyme. Our failure to ohwrvc specificity is consistSent, with the rwulb ol)tainrd hy S. L. Brown, C. A. Hutvhinson I1 1 and M. Smith (unpuhlishrad olwrvatjions). ~4io find t,hat J!boll rwcynizw the sequence 5’G-A-A-G-A 3’. \vith t,hc site of cleavage eight nucleotidw ;\\\‘a>. from thv 3’-terminal nurlrotidc. Since t tit\ c~ompletion of this work, n-e haw twrnc~d that I)pnl 1 from Z)i~~lococcu.~ prw7~n~m irw (Lacks & (bvenberg, 1975) and Sn/tS.-\ 1. from Staphlococcus anew SA (Sussellt)i\cll it NZ.. 1!)7A) have a specificit> id(itltici\t \I it,h that of ~Whol. >C’CI”“‘l”~‘.

1)X.\ samples (0.5 t,o 2. 0 pg) W’PPP incrtbatcti with 0.1 t o I.0 unrts ~~nd~u~r~~lt~asr for 15 h and fractir)nat,wl on 1.4”, agarosr~ slab gc+ as I)NA; 3: MboI Fig. I. Slot 1, MboI on lambda IjK.4 : 2: il601 on Ad-2 011 lambda 1)N.k: 5: Mb011 on Ad-2 DNA; 6: AlboIl on SV40 I)NA: X: .llhoI and Mosl on lambda DNA: 9: Mosl on lambda I)NA; 10: MboI and MonI on Ad-2 DNA; 12: MosI on Ad-2 1)NA: 13: MhoI on SV40 OII SV40 1)NA; 15: MosI on 8V40 1)N.A; 16: UnmHI on SV40 I)NA; SV40 I)NA; 18: Mb01 on XV40 DS.4: 19: Hnn~Hl on Ad:’ 1)N.l; 20: I)KA: 21: MboI on Ad-” DNA: 22: &$I1 on .itl-2 I)S.\: “3: &$I1 24: Jf601 on AtII)NA.

of the indicated type II dwcribed in the legend tl) on RV40 DNA: 4: Mb011 7: MhoI rm lambda DNA: on Ad-2 DNA; 11: Mb01 D?jA; 14: MboI and Most 17: BtrmHI and Mb01 011 KumHL and Mb01 on Ad-:! a11r1 .WboI on Ad-t’ I)XA:

FIG. 5. Pancreatic 1)Sa.w digestion products of 5’.terminally labeled JlboI and 1ActtlHI fragments. Hyphens have been omitted for &wit\ (a) 5’,‘-terminal oligonucleutidc~ from HuntHI fragments were obtained and identified as described previously (Itobcrts et trl., 1976b). (b) 5’.terminal oligonuclrotide~ from XboI fragmenrs were obtained and identified using a lwocedurr sin&r to that described previously. RnmHl fragments (Roberts C/ c/l.. lRi6b). Fractionatiolr in the wrond dimension used thv +ame h~m~umis (number VI: 3a.y rf (II., 1974) but for time.

for tht, a Aorrw

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EDITOR

179

This study was supported by grants from the National and the National Science Foundation (GB43912). One author Public Health research grant no. CA01262 from the National Cold (‘old Krcrivrtl

Spring Spring

Harbor Harbor,

Laboratory N.Y. 11724,

24 December

1976,

and

Cancer Institute (CA13106) (R. E. G.) was supported byCancer Institute.

RICHARD E. GELINAS PHYLLIS A. MYERS RIPHARI) .J. ROBERTS

U.S.A. in revised

form

2 March

19i7

REFERENCES Aaij, c’. & Borst, P. (1972). Biochim. Biophys. Acta, 269. 1922200. Brownlee, G. G. & Sanger, F. (1969). Eur. J. Biochem. 11, 395-399. Glynn. I. M. & Chappell, J. B. (1964). Biochem. J. 90. 147 -149. *Jay. E., Bambara, R., Padmanabban, R. & Wu, R. (1974). Nucl. dcids Res. 1. 331-353. Lacks, S. & Greenberg, B. (1975). J. Biol. Chem. 250, 4060.--4066. Marinus, M. G. & Morris, N. R. (1975). Mutat. Res. 28. 15-26. Pirrotta, V. (1976). Nucl. Acids Res. 3, 174771760. Richardson, C. C. (1965). Proc. Nat. Acad. Sci.. C.S.A. 54, 158165. Roberts, R. J. (1976). CRC Crit. Rev. Biochem. 4, 123. 164. .Robert,s, R. J . . Myers, P. A., Morrison, A. & Murray, K. (1976a). J. Mol. Biol. 102, 157.-165. Roberts, R. J., Wilson, G. A. & Young, F. E. (1976h). ,Vature (London), 265, 82-84. Shalt,irl, S. & Er-El, Z. (1973). Proc. Nat. Acad. Sci.. G.S.A. 70, 778-781. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemistry, 12, 3055-3063. Sugden, B., DeTroy, B., Roberts, R,. J. & Sambrook, .J. (1975). Anal. Biochem. 68. 36-46. Sussenbacb, J. S., Monfoort, C. H., Scbiphof, R. & Stobberingb, E. E. (1976). Xwd. dcids Ren. 3, 3193-3202. Taylor, J. F. (1953). In The Proteins (Neuratli, H. & Bailey. K., eds). 1st edit., vol. 1. part A, pp. 5455, Academic Press, Nru York.