.J. AIlol. Bid.
(1976) 102, 1577165
LETTERS TO THE EDITOR
A Specific Endonuclease from Arthrobacter luteus A new restriction-like endonuclease, AZuI, has been partially purified from Arthrobacter luteus. This enzyme cleaves bacteriophage X DNA, adenovirus-2 DNA and simian virus 40 DNA at many sites including all sites cleaved by the cndonuclease Hind111 from Haernophilus in$uenzae serotype d. Radioactive oligonucleotides in pancreatic DNAase digests of (5’-32P)-labelled fragments of phage X DNA released by the action of AZuI had the 5’ terminal sequence PC-T-N-. The enzyme recognises the tetranucleotide sequence 3’ J&-A5’ 5’ -A-G-C-T- 3’ and cleaves it at the position marked by the arrows. Studies of DNA structure and function are being greatly aided by the use of bacterial restriction endonucleases, many of which both recognize and cleave a specific sequence of base-pairs within a DNA duplex. Several such specific endonucleases have been reported (Middleton et al., 1972; Roberts et al., 1975; Sharp et al., 1973; Smith & Wilcox, 1970; Takanami, 1973; Takanami & Kojo. 1973; Yoshimori, 1971) showing a range of different sequence specificities. Their detection and isolation is facilitated by virtue of a simple assay procedure using agarose slab gel electrophoresis of DNA digests (Sharp et al., 1973; Sugden et al., 1975), which gives distinctive patterns of bands with these specific endonucleases. We report here the isola’tion of a new specific endonuclease, AM, from Arthrobacter luteust. broth (Difco) and cultures were d. hteus, ATCC 21606, was grown on nutrient harvested in a stationary phase. The cells (5 g) were disrupted by sonication in two volumes of a buffer containing 0.01 M-Tris.HCl (pH 74), 0.01 M-%mercaptoethanol. After high-speed centrifugation (100,000 g for 90 min) the supernatant fluid was made 1.0 M in NaCl and DNA was removed by fractionation on a column (50 cm x 2 cm diameter) of Bio-Gel A 0.5 m (Biorad), which was eluted with the same buffer containing 1-O ivf-NaCl. All fractions were assayed for nucleases by agarose gel electrophorrsis of DNA digests and those containing exonucleolytio or endonucleolytic activity were combined and dialysed against a buffer containing 0.01 M-potassium phosphate (pH 7*4), 0.01 M-2-mercaptoethanol, 0.0001 M-EDTA(Na,), 10% glycerol (phosphocellulose buffer) and applied to a column (25 cm x 1.2 cm diameter) of phosphocellulose (Whatman, Pll) previously equilibrated with the same buffer. Elution was carried out with 200 ml (total volume) of a linear gradient from 0 to 1-O MKC1 in phosphocellulose buffer. The assay of fractions from this column is shown in Figure l(a). Endonuclease AluT eluted between O%O and 0.75 M-KCl. Peak fractions were judged to be essentially free of contaminating nucleolytic activity because DNA t Although it hea not been demonstrated that the endonuclease AZuI is in fact a restriction enzyme. we have used for its description, the abbreviation nwthod suggested by Smith & Nathan* (1973) fix restriction and modification enzymes. 167
I
4
I’
lc7
13
16
19
22
25
20
30
32
34
36
38
4C
4?
fiS
46
48
ultraviolet. light on panchromatic film with a red filter. Fraction 11 was being collected. (b) Assay of fractions from the DEAE-cellulose column. Portions (2 ~1) of column fractions were assayed as described each channel in the gel. The gradient was started while fraction
(b)
above
Fraction
in t.he gel. The gradient
was for 2 h at Yi'C.
each channel
to Fig. 1. Digestion collected.
are indicated
in the legend 14 u-as being
numbers
numbers
was
ar ‘c imlicated
start ccl u hile
above
fl *action
gel rlect~toph(~lr\-;la of ~~n(l~,t~~~cl~:a;t~ .4/1tl atlcl Hi~t~llII (ligLt&s of A. All-2 H.llll FIG . 2. Agaroso sv40 DNAs. mlxt,uro contain] 1% Eat :h DNA sample (2 rg) was d~gost~od at 37 ,(.’ t’ur I ti h ~11u 50.~1 ruactu)n 0 mzr -“-rnc~rcaptopthan[)l, and 0.1 units of t,he indical :ed (pH 7.9). 6 mix-Mg:C’l,, 6 mM -Tris.HC!I (1 to 5 ~1). Digest,s WHI’H fract,iorrnt,od as described in t,he legend to Fig. 1. specif io endonuclease UII X DNA; slot 3, Hind111 on X DNA; slot 4, slot 2, AZuI + Hind111 Slot’ 1 , AZuI on X DNA; slot 5, AZuI -k R.ZlinclIIT on Ad-2 DNA; slot 6, R.HindJII on Ad-2 DN ‘A; AZuI on Ad-2 DNA; slot 8, Ah1 + R.HindIII on SV40 DNA; slot 9, H.WindIII on SF ‘40 slot, 7:, AZuI on SV40 DNA; digest of SV40 DI\;A in channel 9 was incomplet,e because snpwcoiletl SV ‘40 DNA. The R.HindIII for R.HindIII. DNA Iis a poor substrate
Cl0 Cl1
(‘7 68
C9
Cl C2 03 C4 c5 C6
0.22 0.14 0.15 0.10 0.28 0.25 0.13 0.19 0.17
1.11 0.86 0.52 0.40 0.36 0.31
Mobility$ (AE, pH 3.5)
I.23 1.01 0.90 0.81 0.78 0.67 0.62 0.21 0.47
2.60 2.16 1.83 0.77 1.43 1.58
Mobilityt (DE, pH
.fragments
2.0)
PC PC PC PC’
PC PC pC Jf
pc
PC PC PC
PC pc
PC
5' Terminus
generated
PC-T, PC-T, PC-T, PC-T, J&-T. PC-T, PC-T, PC-T, PC-T,
pc-T PC-T PC-T PC-T,
Partial
diyestiotl~
PC-T-C, PC-T-G PC-T-I PC-T-T,
PC-T-C PC-T-A, PC-T-A PC-T-A,
PC-T-C
digestion
PC-T-:1
PC’.T-T
I-‘(‘-T-A
PC.‘-T-.&C -( ’
producta§
XluI
PC’-T-T--k
J,C:-T-T-T
p(:-T-CA PC’-T-A-C-C PC-T-A-A PC’-T-A-C-A @-T-G PC-T-C-T PC-T-G-A
PC’ PC-T PC-T-C’ PC-T-T PC-T-A pC-T-C-C’
of 5’ terttritlall,t/
of ett,donuclease
DXAase
by the action
obtaitded by pancreatic
of h DXA
and
and
Itlentitj
PC-T-A-T
PC’-T-T-(’
t The oligonucleot,ides are identifirtl in Fig. 3, lvttcrs referring to t,hr separation syst,em, and numbers tu t,he oligonucleotide m that system. : Mobilities are with respect to the blue tlyv , xylent: cyan01 FE’. IThere the same oligonucleotide was isolated on two fractionation systems, duplicate values were obtained and have been averaged. § Oligonucleotides in partial digests with venom phosphodiesterasr (0.01 mg enzyme/ml in 0.02 %r-Tris.HCl (pH 8.5), containing carrier oligonucleotides--DNAasc digest of calf thymus DSA---50 pg/ml at 23”C, samplod at 15 min and 00 min). Digests of nuclcotides shown in Fig. 3 (a) and (c) were analyscd on DE81 paper and those from Pig. 3(b) on AE 81 paper (Murray, 1973).
B7, J% B9, 810, Bll B12: B13, B14,
Al, -12, -43, 84, A5,
Oligonucleotide numbert
labelled
A tt~alysis of the oligormcleofides
I+
Cb)
.
t I I
1
+4 fgCl, a,t 37°C for 135 min (Fig. 3(c)). The first digest was fractionated by ionophoresis on AE8 1 paper at p.H 3.5 (and part, of this was used for the second dimension ionophoresis on DE81 paper at pH 2.0) and the second by 2dimensional ionophoresis on cellulose acetate and AE81 paper, both at pH 3.5. Oligonucleotides were located by radioautography and eluted wkh 309;, tri~,tllvl;11llrlrorlillln hicarbonate for analysis by dipcstion with snalw venom I’hosphotliesterase.
I64
I, : &wnI (Wilson & Young. 1975), HapI, Hapll. HapIll. Nha. Hindll. H&dlll, H/JuI. EI~KLZI. EcoRI, EcoRIl (the enzymes are as dt~scril)td 1)y Natllans & smith. 1975). In all cases but one, BZuI had a clear1.v different, sequmw specificity as judged 1,~.thtn appearance of additional bands in the double digest compared with either single digest. The exception was R.HindlI I. one of the rndonucleases from Huemophi2u.s of hands obtained from t,he double in@uenzae serotype d. In this case the pattern digest was identical with that from the AZuI digest alone, indicating that the sites of cleavage by R.HindIlI were a subset of the sites cleaved by dlul. The sequent recognized by RX&d111 is 5’ -A-A-G-C-T-T3’ (Murray $ Old. 1974; Old it (11.. 1975) and it was, therefore. tempting to suggest that the cacntral t,etranllcl(,otitl(, -A-G-C-T- is the recognition sequence for Blul. Direct proof of the recognition sequence and determination of the positions of breakage by the enzyme come from analyses of DN8ase digests of 5’ terminall! labelled reaction products, as described previously (Bigger ef al.. 1973: Old et ~1.. 1975). Fragments of phage X DNA generated by t’he action of &UT \\ere dephosphorylated with bacterial alkaline phosphatase and labelled with 321’ at, their 5’ termini by incubation with polynucleotide kinase and [ y-32P]ATP (Richardson. formed on digestion wit~lr 1965 ; Novogrodsky & Hurwitz, 1966). 01’igonucleotides pancreatic DNAase were fractionated by ionophoresis on Whatman AE81 and DE81 ion-exchange papers and by two-dimensional ionophoresis on cellulose acetate and AE81 paper (Fig. 3). Radioa,ctive oligonucleotides were elut’ed, digested (partially) with venom phosphodiesterase and their nucleotide sequences deduced from the behaviour of the digestion products on ionophoresis on Whatman BE81 and DE81 ion-exchange papers (Murray, 1973). The results, which are summarized in Table 1. showed that all the oligonucleotides had the 5’ terminal dinucleotide C-T-. Taken together with the electrophoretic analyses of double digests of DNA with cndo R.HindIII and endo BZuI and the known recognition sequence for R.HindILi (Old et al., 1975), this result establishes that BZuI recognises the tetranucleotidc sequence -A-G-C-T- and makes an even break in double-stranded DNA molecule.; through the middle of this sequence (and its complement). We have not determined whether cleavage of the phosphodiest,er bond leaves a 5’ phosphate or a 5’ hydroxyl group. We do not know whether AU is involved in a host-controlled restriction and modification system or not. This investigation was supported by a grant (CA13106) from tbo National tute and by the Science Research Council (U.K.).
Cancer InstiJ.
Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y. 11724, U.S.A.
RICHARD
ROBERTS
Department Edinburgh, Scotland
ALAN MORRISONt KENNETH MURRAY
PHYLLIS A. MYERS
of Molecular Biology, University of King’s Buildings, Edinburgh EH9 3JR,
Received 31 July 1975 t Present address: Department Scotland.
of Biochemistry,
University
of Aberdeen,
Aberdeen
AB9 IAS,
REFERENCES Bigger, (1. H., Murray, K. & Murray, N. E. (1973). Sat/we Xew Did. 244, i-10. Clausen, T. (1967). Anal. &o&em. 22, 70-73. Middleton, J. H., Edgell, M. H. & Hutchison. (‘. A. III (195”). ./. I.iro/. 10, 42--W. Murray, K. (1970). Biochem. J. 118, 831-841. Murray, K. (1973). Biochem. J. 131. 560 583. Mnrra~~. K. dt Old, K. IV. ( 1974). f’rog. Nwleic Acid Kes. Xol. Hiol. 14. 117 -185. Nathans, D. & Smith. H. 0. (1975). Bnnll. Rev. Rio&em. 44, %73&,“93. Novogrodsky, A. bt Hurmitz. ,J. (IQBG). J. Bid. Chew. 241, XQ23- 203%. Old, K. W., Murray. K. & Roizes. G. (1975). ./. ,IIo/. Wlol. 92. 331 339. Richardson, C. C. (1965). Proc. A’at. Acad. Sci.. I’.S.il. 54, 158 IfiR. Hobcrts. K. .I., Rwitrrmyer. .J. H.. Tahacllnik. N. F. & Mc>.c>rs. I’. .-I. (1!)75). .I. ,210/. Hiol. 91. 121- 124. Sharp, 1’. A.. Supden, B. 62 Sambrook, .J. (1Q73). HiockemGtry. 12. 3055 3063. Smitlr, H. 0. & Nathans, D. (IQ73). J. Mol. Riol. 81, 419 423. Smith. H. 0. & \Vilcox, K. 11:. (lQ70). J. &IO/. Biol. 57. 37%31)1. Sugdell. B., DeTroy, B., Kobcrts, K. J. $ Sambrook, .I. (lQ75). .dnn/. Biochem. 68, :jBk#i. Wilson. G. A. & Young, F. E. (1!)75). J. ,Ilol. Biol. 97. 123 l&j. Takanami, M. (1!,73). PEBS Letters. 34, 318~ 322. Takanami, M. & Ko,jo, H. (lQ73). FEBS Letters, 29: “67 2i(). Yosltirtlori. H. N. (I 971). Ph.D. Tllcsis. Univtwity of (‘alifortlia.
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