J. Biochem. 86, 627-637 (1979)
for Single-Stranded DNA from Bacillus subtilis Marburg Kouji MATSUMOTO,1- • Tadahiko ANDO,* Hiuga SA1TO,** and Yonosuke IKEDA** *The Institute of Physical and Chemical Research, Hirosawa, Wako, Saitama 351, and "The Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, October 5, 1978
Bacillus subtilis Marburg TI (thy.trpCt) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular f 1 DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endonuclease Mil), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57,000 daltons. The cleavage products have 5'-phosphoryl termini. At low concentrations, double-stranded DNA is not split to any detectable extent. At high concentrations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5,000 : 1 by comparing the initial rates of introducing single-strand breaks into the DNA's.
Nucleolytic enzymes which is specific for singlestranded DNA have been purified and extensively studied in fungi and plants (.1-8). The enzymes isolated from these organisms are used mainly in the study of DNA structure and in genetic manipulation. In bacteria, few studies on similar enzymes have appeared (9-12). These enzymes are considered to play important roles in gene conversion and genetic recombination (3,13,14). The 1
K.M. is on leave from The Institute of Applied Microbiology, The University of Tokyo. Communications should be addressed to K.M. at the Life Science Institute, Sophia University, Kioi-cho, Chiyoda-ku, Tokyo 102. Vol. 86, No. 3, 1979
627
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Purification and Characterization of an Endonuclease Specific
hypothesis that a single-stranded DNA-specific nuclease of Ustilago maydis acts on mismatched DNA, which is presumably formed during these processes, has obtained experimental support (75, IS). We have reported that the efficiency of genetic transformation in Bacillus subtilis Marburg by DNA of hybrid strains carrying the marker of B. amyloliquefaciens 203 strain is enhanced considerably by a coexisting mutation in the recipient strain (17). One possible hypothesis to explain the phenomenon is that the wild-type strain might have some endonuclease activity which is specifically able to attack mismatched or single-stranded portions on the donor-recipient DNA complex. We were therefore led to the study of single-
628
K. MATSUMOTO, T. ANDO, H. SA1TO, and Y. IKEDA
stranded DNA-specific endonuclease in Bacillus sublilis Marburg. This paper describes the purification and properties of the enzyme.
/. Biochem.
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assay mixture contained 50 mM glycine-NaOH (pH 8.8 at 37°C), 5 mM MnCl,, 2 mM 2-mercaptoethanol, 0.4 fig of sH-labelled fl DNA (7,100 cpm/fig), and 10% glycerol in a total volume of 25 ft\. After incubation for 20 min at 37°C, the MATERIALS AND METHODS reaction was stopped by adding 5 fi\ of SEBP Bacterial Strains and Phages—TI (thy, solution (40% sucrose, 0.1 M EDTA, 0.05% trpCj') strain of Bacillus subtilis Marburg was bromphenol blue, and 5% phenol). The samples provided by Dr. F. Rothman. Phage SPPl and were subjected to electrophoresis on vertical 1.6% B. subtilis MCB were provided by Dr. H. Hirokawa. agarose slab gels at 110 V for three h in the absence Phage fl and E. coli W2252 (F~, X~, met") were of ethidium bromide. The top band and the from Dr. E. Hayase. bottom band are single-stranded circular DNA and whole linear DNA, respectively (22). Each Enzymes—DNase 1 of bovine pancrease (Worthington), alkaline phosphatase of E. coli DNA band was cut out and the radioactivity was (Boehringer), and micrococcal nuclease (Worthing- measured by using toluene scintillator in a Beckman ton) were purchased. T4 polynucleotide kinase LS-230 liquid scintillation system. On quenching, (phosphocellulose fraction, a kind gift of Dr. S. 43% of the input radioactivity was counted by Kitayama) was further purified on Ultrogel AcA34 this procedure. The relationship between enzyme according to the method described by Panet et al. concentration and enzyme activity is shown in Figs. 1 and 2. A roughly linear relationship was (18). Chemicals—Ultrogel AcA34 (LKB), DEAE- obtained up to about 40% conversion of the cellulose DE52 (Whatman), denatured DNA- single-stranded circular DNA to the linear form. cellulose (Wako Pure Chem. Co.), [r-"P]-ATP One unit of the enzyme activity was defined as (New England Nuclear), [6-8H]-thymidine (Radio- the amount of the enzyme which converts 0.1 fig chemical Centre, Amersham), Ficoll (Pharmacia), of single-stranded circular fl DNA to the linear bovine serum albumin (Schwartz), ovalbumin form under the standard reaction conditions with (Schwartz), and myoglobin (Schwartz) were 0.4 fig of single-stranded circular f 1 DNA as a substrate, (b) The endonuclease activity for purchased from the indicated sources. DNA and RNA—»H-Labelled phage f 1 DNA (7,100 cpm/^g) was prepared as described by a b Marvin and Schaller (19). »H-Labelled fl doublestranded superhelical DNA (replicative form 1 DNA) was prepared as described by Model and Zinder (20). The final step of the purification was ethidium bromide/CsCl density gradient centrifugation. The specific radioactivity was 33,000 cpm//ig. Cold and 3H-labelled SPPl DNA circular were prepared as described by Riva et al. (21). S The specific radioactivity of the H-Iabelled SPPl linear DNA was 18,000 cpm//ig. Ribosomal RNA of B. subtilis was the kind gift of Dr. H. Takahashi. Assays of Endonuclease Activity—(a) The endonuclease activity for single-stranded DNA was assayed by measuring the conversion of singlestranded circular DNA of phage fl to the linear Fig. 1. Dose-response relation for endonuclease MIL Various amounts of enzyme diluted to about 1,000 form. This method is based on our finding that units/ml (Fraction V) were added to 25 ft] of the standsingle-stranded circular DNA and whole linear ard reaction mixture. Assays were conducted as deDNA can easily be separated by electrophoresis on scribed in " MATERIALS AND METHODS." Slots 1.6% agarose gel. The details of the method are a through g are the digestion products with 0, 0.5, 1, described in a separate paper (22). The standard 1.5, 2, 4, and 6 ft\ of Mil, respectively.
SINGLE-STRANDED DNA-SPECIFIC ENDONUCLEASE OF B. subtilis
0
1 2
4
S
ENDONUCLEASE M I K J J I )
Fig. 2. Dose-response curve for endonuclease MEL The radioactivity of each top-band (single-stranded circular DNA) of Fig. 1 was measured as described in " MATERIALS AND METHODS." The decreases in radioactivity of circular DNA in the reaction mixture are shown. double-stranded superhelical DNA was determined by measuring the conversion of 'H-labelled doublestranded superhelical DNA of f 1 to open-circular and linear DNA's by neutral and alkaline sucrose gradient sedimentations and by agarose gel electrophoresis. As these products form discrete bands on 0.7% agarose gel electrophoresis, the radioactivity of each DNA band could be measured. The reaction was carried out in the standard reaction mixture supplemented with NaQ to give a final concentration of 120 mM. (c) The nuclease activity for double-stranded linear DNA was assayed with SPP1 DNA. The DNA samples were analyzed qualitatively by 0.7% agarose gel electrophoresis and quantitatively by alkaline sucrose gradient sedimentation.
the presence of 0.5 /ig/ml of ethidium bromide. The 1.6% gel was stained after electrophoresis. DNA was visualized under ultraviolet light and photographed with Tri-X film (Kodak). To determine the radioactivity of each DNA band, the band was cut out from the gel plate and the agarose was hydrolyzed with ca. 0.1 N HC1. Sucrose Gradient Sedimentation—Neutral and alkaline sucrose gradient sedimentations of digestion products of f 1 DNA were done at 4°C in the Hitachi RPS65T rotor at 50,000 rpm. Neutral 5-20% sucrose gradients (4.7 ml) containing 0.9 M NaCl, 10 mM Tris-HCl, pH 8.0, and 5 mM EDTA were centrifuged for 180 min (single-stranded circular DNA) or for 240 min (double-stranded superhelical DNA), and alkaline sucrose gradients containing 0.25 N NaOH, 0.7 M NaCl, and 5 mM EDTA were centrifuged for 120 min (doublestranded superhelical DNA). Alkaline sucrose gradient sedimentation of digestion products of SPP1 DNA was carried out in the Hitachi RPS40T2 rotor at 38,000 rpm for 180 min. Two-drop fractions were collected directly onto GB100R glassfilters(Toyo Roshi Co. Ltd.) from the bottom of the polyethylene tube. The radioactivity was measured as described previously. The molecular weight of DNA was estimated according to the method of Abelson and Thomas (23).
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400
629
Glycerol Gradient Sedimentation—Sedimentation of endonuclease Mil was done at 4°C in the Hitachi RPS65T rotor at 44,000 rpm for 14 h. Gradients of 10-30% glycerol containing 20 mM Tris-HCl, pH 7.5, and 2 mM 2-mercaptoethanol were used. Fraction V of the enzyme was dialyzed against buffer B containing 7% glycerol prior to centrifugation. After centrifugation, two-drop fractions were collected from the bottom and the Agarose Gel Electrophoresis—The apparatus endonuclease activity was assayed by the standard for agarose slab gel electrophoresis was the vertical procedure. model of Studier (24) with minor modifications. Polynucleotide Kinase Treatment—PhosphoAgarose gels were prepared by dissolving agarose rylation of the endonuclease-digested DNA by (0.7% or 1.6% w/v, Sigma) in TAE buffer (40 mM T4 polynucleotide kinase followed the method Tris-acetate, 5 mM sodium acetate, 1 mM EDTA, described by Panet et al. (18). The reaction pH 7.8) and poured into a mold between two glass mixture (100 p\) contained 50 mM Tris-HCl (pH plates (140x120x4 mm). Samples were placed 8.0), 10 mM MgCl,, 5 mM 2-mercaptoethanol, in each well on the slab gel after being mixed with 2 mM KH,PO4, 2 mM spermine, 4 ftg of DNA, one-fifth volume of SEBP solution- Electro- 100 (iM [T-"P]-ATP (114,000 cpm), and 0.2 unit phoresis was carried out at 110 V at room tem- of T4 polynucleotide kinase (Ultrogel AcA34 perature for 2-3 h in TAE buffer. In the case filtrate fraction). The reaction mixture was incuof the 0.7 % gel, electrophoresis was carried out in bated at 37°C for 30 min. Vol. 86, No. 3, 1979
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K. MATSUMOTO, T. ANDO, H. SATTO, and Y. DCEDA
RESULTS
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Purification of Endonuclease Mil—Preparation of extract: TI strain of B. subtilis Marburg was grown in 6 liters of tryptone yeast extract medium and collected at the early stationary phase by centrifugation. The cells (20 g) were suspended in 70 ml of Buffer B (20 HIM Tris-HCl pH 7.5, 2 rrun 2-mercaptoethanol, 10% glycerol) containing 10 mM 2-mercaptoethanol and were treated with 50 /ig/ml of lysozyme at 37°C for 30 min. The protoplasts produced were subjected to sonic oscillation (4 times for 30 s each at a setting of 4.5) with an ultrasonic disruptor (UR-200P, Tomy Seiko Co.). 30 40 50 60 The cell debris was removed by centrifugation in FRACTION WJMBER the Hitachi RP30 rotor at 28,000 rpm for 90 min. Fig. 3. DEAE-cellulose chromatography of Fraction The supernatant (69 ml) was collected and desig- II. Endonuclease activity towards single-stranded cirnated as Fraction I. cular DNA was assayed in the standard reaction Ammonium sulfate precipitation: Powdered mixture. Endonuclease activity (•); protein concenammonium sulfate (21.3 g) was added to fraction tration measured as absorbance at 280 nm (O); molarity ). I with stirring. After further stirring for 30 min, of KCl ( the precipitate was removed by centrifugation. Powdered ammonium sulfate (19.8 g) was added also contained activity towards single-stranded slowly to the supernatant (79 ml) with stirring for DNA and towards double-stranded DNA in the 30 min. The precipitate was collected by centrifu- presence of CaClj or MgCla (data not shown). gation and dissolved in Buffer B (Fraction n, The fraction at 0.17-0.19 M KCl (tube Nos. 45-47) 50 ml). was pooled and concentrated to 11 ml using Ficoll DEAE-cellulose chromatography: Fraction II (Fraction HI). was dialyzed against Buffer B and applied to a Gel filtration: The concentrated Fraction i n DEAE-cellulose column (2.5x11 cm) previously was loaded on an Ultrogel AcA 34 column (2.5 x equilibrated with Buffer B. The column was 44 cm) previously equilibrated with Buffer B washed with the buffer and eluted with a linear (supplemented with 0.1 M KCl) and washed with a gradient of 0 to 0.3 M KC1 in Buffer B (300 ml) small volume of the buffer. Elution was carried at the flow rate of 17ml/h; fractions of 7 ml were out with the same buffer at the flow rate of 15 ml/h collected. Figure 3 shows the distribution pattern and fractions of 4.9 ml were collected. Figure of endonuclease activity for single-stranded DNA. 4 shows the distribution pattern of the endonuclease The nuclease activity for double-stranded DNA activity. The fractions exhibiting high activity was also assayed qualitatively with double-stranded towards single-stranded DNA but no activity on linear DNA of SPP1 under the same reaction con- double-stranded DNA (tube Nos. 29-32) were ditions by means of agarose gel electrophoresis. pooled (Fraction IV, 19.6 ml). Among the three peaks of activity towards singleThe Second DEAE-cellulose chromatography: stranded DNA eluted from the column, the fraction Fraction IV was dialyzed against Buffer B and at 0.17-0.19 M KC1 exhibited no activity on double- loaded onto a DEAE-cellulose column (2.5 x 3 cm) stranded DNA. However, the fraction eluted at previously equilibrated with Buffer B (supplemented 0.1-0.14 M KC1 exhibited high activity towards with 0.1 M KCl). The column was washed with double-stranded DNA in the presence of MnCli the buffer and the enzyme was eluted with 300 ml or CaClj. The fraction at 0.2-0.25 M KC1 ex- of a linear gradient of 0.1 to 0.3 M KCl in Buffer hibited enhanced activity for double-stranded DNA B. Theflowrate was 30 ml/h, and 3.5 ml fractions in the presence of ATP. The unadsorbed fraction were collected. The peak of the enzyme activity
SINGLE-STRANDED DNA-SPECIFIC ENDONUCLEASE OF B. subtilis
20
30
40
SO
FRACTION NUMBER
Fig. 4. Gel filtration of Fraction III on Ultrogel AcA34. Endonuclease activity towards single-stranded circular DNA was assayed in the standard reaction mixture. Endonuclease activity ( • ) ; protein concentration measured as absorbance at 280 nm (O).
30 ml of a 0.05 to 1.0 M KC1 gradient. The flow rate was 4.5 ml/h, and 1 ml fractions were collected. The fractions which exhibited high activity (eluted at 0.3-0.45 M KC1) were pooled (Fraction VI, 5 ml). DEAE-celluIose concentration: Four ml of Fraction VI was dialyzed against Buffer B and adsorbed onto a DEAE-cellulose column (0.5 x 1.2 cm) previously equilibrated with Buffer B (containing 20% glycerol). The enzyme was eluted with the same buffer containing 0.25 M K G . Fractions of 0.6 ml were collected and the most active fraction was used in the present experiments (Fraction VII, 0.6 ml). These fractions (V, VL, and VII) were supplemented with glycerol (final concentration, 50%) and stocked at - 2 0 ° C . The purification procedure is summarized in Table I. The enzyme was purified more than 320 times on a specific activity basis. Properties of Endonuclease Mil—Stability: The enzyme was labile after the second DEAEcellulose chromatography. Addition of ethylene glycol or glycerol stabilized the enzyme activity. The enzyme retained more than 90 % of its original activity in Buffer B containing 50% glycerol after storage for five months at —20°C. Optimal pH for action: The activity versus pH relation was assayed in a reaction mixture which contained 5 mM MnClj. The optimal p H was found to be between pH 8 and 9,40% and 10% of the optimal activity being observed at pH 7 and pH 10, respectively.
TABLE I. Purification of endonuclease Mil. The activity was assayed under the standard reaction conditions. Protein concentration was measured according to the method described by Lowry et al. (25). Fraction I. Crude extract II. Ammonium sulfate III. IV. V. VI. VII.
DEAE-cellulose I AcA 34 filtration DEAE-cellulose 11 DNA-cellulose* DEAE-cellulose concentration
Total protein (mg)
Total activity (units XlO"')
Specific activity (units xlO-'/ m 8 protein)
4,026 1,410 338 32.9 3.19 0.228 0.16
9,662 8,800 3,360 1,848 504 175 123
2.4 6.2 9.2 56.2 158 768 770
* Only a portion of Fraction V was used for further purification by denatured DNA-cellulose chromatography; the data presented were recalculated accordingly. Vol. 86, No. 3, 1979
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appeared at 0.17-0.19 M KC1. At this purification step, the fraction was apparently free from nuclease activity towards double-stranded DNA, even on prolonged incubation. The peak fractions were pooled (Fraction V, 14 ml). Denatured DNA-cellulose chromatography: Three ml of Fraction V was dialyzed extensively against Buffer C (20 mM Tris-HCl, pH 7.5, 2 rriM 2-mercaptoethanol, 1 mM EDTA, 50 mM KC1, and 10% glycerol) and loaded onto a denatured DNAcellulose column (0.8x2.8 cm) equilibrated with Buffer C previously. The column was washed with Buffer C and the enzyme was eluted with
631
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K. MATSUMOTO, T. ANDO, H. SA1TO, and Y. IKEDA 1 mM and 25 mM potassium phosphate, respectively. Effect of RNA: When the enzyme was assayed in the presence of 2.1 or 4.2 fig per 25 pi of B. subtilis ribosomal RNA, the activity was inhibited by 35 % or 65 %, respectively. Absence of exonuclease activity: Singlestranded circular f 1 DNA was treated with 5, 10, and 20 units of the enzyme (Fraction V) and the digestion products were analyzed by sucrose gradient sedimentation. The absence of exonuclease activity was confirmed by the absence of nonsedimentable material (data not shown; refer to Figs. 7 and 10). Absence of phosphatase activity: The enzyme had no phosphomonoesterase activity, as indicated by the negligible formation of p-nitrophenol in the standard reaction mixture and in 1 M Tris-HCl buffer (pH 8.0) containing 1 mM p-nitrophenyl phosphate and 200 units of the enzyme (fraction V). Molecular weight: The size of the endonuclease was estimated by the gel filtration method according to Andrews (26). The estimated molecular size of Mil protein was 57,000 daltons as shown in Fig. 6. Glycerol gradient centrifugation of the enzyme was also carried out, and a Svedberg value of 4.2 was obtained by comparison with bovine serum albumin. Substrate Specificity of the Enzyme—Absence of action on double-stranded linear DNA: Throughout the purification procedure the endo-
20
T4 potynucleotid* klnase
"10 x 8
Ineseum albunin
I:
Mil
ovalbumln
myoglobln 1.2 2
5
10
20
50
( mM ) Fig. 5. Effects of divalent cations on the activity. MnCl, (5 mM) in the standard reaction mixture was replaced by the indicated concentrations of CaCl- (•), MnCl, (O), and MgCl, (•). Each activity was expressed as a percentage of the activity obtained with 5 mM MnCl,. CATION
1-4
16 Vtf Vo
18
2-0
Fig. 6. Estimation of molecular size by gel filtration. MH was loaded on an Ultrogel AcA34 column (1 x 52 cm) and eluted with Buffer B containing 0.1 M NaCl. The following proteins were used as standards: T4 polynucleotide kinase, M.W. 140,000 (18); bovine serum albumin, M.W. 67,000; ovalbumin, M.W. 45,000; myoglobin, M.W. 17,800. /. Biochenu
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Effect of divalent cations on the activity: Figure 5 shows plots of relative activity versus divalent cation concentration. CaCl, was the most effective (at 2-50 mM). The optimal concentrations for MnCl, and MgQ, were 2-5 mM and 5-20 mM, respectively. The activity was quite low in the absence of these cations. High concentrations of CaClt, although this was the most effective cation, often prevent the penetration of single-stranded DNA into 1.6% agarose gel in the presence of high concentrations of protein. Therefore, MnClt was used for the standard assay system. Effect of NaCl on the activity: The activity was not inhibited by the addition of 120 mM or 200 mM NaCl under the standard reaction conditions. Even at 500 mM NaCl, about 70% of the activity was still retained. Effect of ATP on the activity: The activity was not affected at all in the presence of 25 or 500 fJM ATP in reaction mixtures in which MnCl, was replaced by MgCla. Double-stranded linear DNA of SPPl was not hydrolyzed by endonuclease Mil (up to 20 units, incubation for 60 min) in the same reaction mixtures when they were tested by 0.7 % agarose gel electrophoresis. Effect of KtHPOt on the activity: Potassium phosphate inhibited the activity strongly. Inhibition reached 80% and 95% in the presence of
SINGLE-STRANDED DNA-SPECIFIC ENDONUCLEASE OF B. subtilis
calculated to be only 0.4 per double-stranded molecule. This result indicates that the endonuclease is highly specific for single-stranded DNA. Action on double-stranded superhelical DNA:
The enzyme (up to 4 units per 25 fi\ for 20 min) did not cleave double-stranded superhelical DNA of fl, as determined by 0.7% agarose gel electrophoresis (data not shown). As it has been reported that double-stranded superhelical DNA is cleaved by large amounts of nuclease SI (27, 28), a similar experiment was performed with high doses of ME using 'H-labelled double-stranded superhelical DNA of fl. To compare the action of MTJ (at high dose) on double-stranded superhelical DNA with that on single-stranded DNA, the time course of cleavage of single-stranded circular fl DNA with 18.2 units of Mil per 25 /il was tested. Within 0.75 min, 37% of the singlestranded circular DNA was cleaved. After two min, 85 % was cleaved, and after 20 min 98 % was cleaved to small fragments (data not shown). Figure 8 shows the cleavage pattern of doublestranded superhelical DNA by 18.2 units per
a b c d e f
oc L DSS
10 20 30 FRACTION (UMBER
Fig. 7. Alkaline sucrose gradient sedimentation of double-stranded linear DNA treated with ME. The standard reaction mixture (150 /il) containing 2.4 fig of s H-labelled double-stranded linear DNA of phage SPP1 (18,000 cpm//ig) was treated with 109 units of Mil. NaCl was added to make a final concentration of 120 IDM. At 0 min (A), after 60 min (B) and after 120 min ( Q of incubation, 50 /il samples were withdrawn, diluted into 150 /il of 5 HIM EDTA solution at 0°C and subjected to sedimentation analysis. Vol. 86, No. 3, 1979
Fig. 8. Time course of cleavage of double-stranded superhelical DNA with MIT. The standard reaction mixture (175 /il) containing 2.8 fig of sH-labelled doublestranded superhelical DNA of fl (33,000 cpm//ig) containing NaCl to give a final concentration of 120 HIM was treated with 127 units of ME. At intervals during incubation at 37°C, 25 fi\ samples were withdrawn and subjected to 0.7% agarose gel electrophoresis. Top, middle, and bottom bands correspond to opencircular, linear, and double-stranded superhelical DNA, respectively. Slots a through / are the digestion products incubated for 0, 10, 20, 40, 60, and 120 min, respectively.
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nuclease activity was assayed in two ways, i.e., with single-stranded circular fl DNA and with double-stranded linear DNA of SPP1 as substrates. As the latter assay was qualitative, the data are not shown. In order to confirm the specificity for single-stranded DNA, double-stranded DNA of SSP1 was treated with higher dose of Mil for longer incubation time and subjected to alkaline sucrose gradient sedimentation analysis (Fig. 7). The molecular size of the DNA treated with 18.2 units of Mil per 25 fi\ for 60min did not change, though after 120 min the molecular size became slightly smaller. The molecular size of the DNA treated for 120 min was estimated to be 10.4 x 10* daltons according to the equation of Abelson and Thomas (23), assuming the molecular size of alkali-denatured SPP1 DNA to be 12.5 x 10* daltons (21). The possible number of nicks introduced after incubation for 120 min was
633
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K. MATSUMOTO, T. ANDO, H. SA1TO, and Y. DCEDA trations of NaCl, while the activity towards singlestranded circular DNA was not inhibited. Marked inhibition of the activity on double-stranded DNA by high concentrations of salts is a common feature of nucleases specific for single-stranded DNA (27-31). These results suggest that ME nicks either strand of double-stranded superhelical DNA at a weakly hydrogen-bonded region and then nicks the remaining strand gradually. Identification of termini of cleaved DNA: The digestion product of single-stranded circular fl DNA with Mil was tested for 5'-termini which would accept 31P from [;--"P]ATP in the presence of T4 polynucleotide kinase, using the method described by Panet et al. (18). Digestion products 2000
cpm
a
for Single-Stranded DNA from Bacillus subtilis Marburg Kouji MATSUMOTO,1- • Tadahiko ANDO,* Hiuga SA1TO,** and Yonosuke IKEDA** *The Institute of Physical and Chemical Research, Hirosawa, Wako, Saitama 351, and "The Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, October 5, 1978
Bacillus subtilis Marburg TI (thy.trpCt) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular f 1 DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endonuclease Mil), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57,000 daltons. The cleavage products have 5'-phosphoryl termini. At low concentrations, double-stranded DNA is not split to any detectable extent. At high concentrations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5,000 : 1 by comparing the initial rates of introducing single-strand breaks into the DNA's.
Nucleolytic enzymes which is specific for singlestranded DNA have been purified and extensively studied in fungi and plants (.1-8). The enzymes isolated from these organisms are used mainly in the study of DNA structure and in genetic manipulation. In bacteria, few studies on similar enzymes have appeared (9-12). These enzymes are considered to play important roles in gene conversion and genetic recombination (3,13,14). The 1
K.M. is on leave from The Institute of Applied Microbiology, The University of Tokyo. Communications should be addressed to K.M. at the Life Science Institute, Sophia University, Kioi-cho, Chiyoda-ku, Tokyo 102. Vol. 86, No. 3, 1979
627
Downloaded from https://academic.oup.com/jb/article-abstract/86/3/627/2185930 by Tulane University Medical Library user on 18 January 2019
Purification and Characterization of an Endonuclease Specific
hypothesis that a single-stranded DNA-specific nuclease of Ustilago maydis acts on mismatched DNA, which is presumably formed during these processes, has obtained experimental support (75, IS). We have reported that the efficiency of genetic transformation in Bacillus subtilis Marburg by DNA of hybrid strains carrying the marker of B. amyloliquefaciens 203 strain is enhanced considerably by a coexisting mutation in the recipient strain (17). One possible hypothesis to explain the phenomenon is that the wild-type strain might have some endonuclease activity which is specifically able to attack mismatched or single-stranded portions on the donor-recipient DNA complex. We were therefore led to the study of single-
628
K. MATSUMOTO, T. ANDO, H. SA1TO, and Y. IKEDA
stranded DNA-specific endonuclease in Bacillus sublilis Marburg. This paper describes the purification and properties of the enzyme.
/. Biochem.
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assay mixture contained 50 mM glycine-NaOH (pH 8.8 at 37°C), 5 mM MnCl,, 2 mM 2-mercaptoethanol, 0.4 fig of sH-labelled fl DNA (7,100 cpm/fig), and 10% glycerol in a total volume of 25 ft\. After incubation for 20 min at 37°C, the MATERIALS AND METHODS reaction was stopped by adding 5 fi\ of SEBP Bacterial Strains and Phages—TI (thy, solution (40% sucrose, 0.1 M EDTA, 0.05% trpCj') strain of Bacillus subtilis Marburg was bromphenol blue, and 5% phenol). The samples provided by Dr. F. Rothman. Phage SPPl and were subjected to electrophoresis on vertical 1.6% B. subtilis MCB were provided by Dr. H. Hirokawa. agarose slab gels at 110 V for three h in the absence Phage fl and E. coli W2252 (F~, X~, met") were of ethidium bromide. The top band and the from Dr. E. Hayase. bottom band are single-stranded circular DNA and whole linear DNA, respectively (22). Each Enzymes—DNase 1 of bovine pancrease (Worthington), alkaline phosphatase of E. coli DNA band was cut out and the radioactivity was (Boehringer), and micrococcal nuclease (Worthing- measured by using toluene scintillator in a Beckman ton) were purchased. T4 polynucleotide kinase LS-230 liquid scintillation system. On quenching, (phosphocellulose fraction, a kind gift of Dr. S. 43% of the input radioactivity was counted by Kitayama) was further purified on Ultrogel AcA34 this procedure. The relationship between enzyme according to the method described by Panet et al. concentration and enzyme activity is shown in Figs. 1 and 2. A roughly linear relationship was (18). Chemicals—Ultrogel AcA34 (LKB), DEAE- obtained up to about 40% conversion of the cellulose DE52 (Whatman), denatured DNA- single-stranded circular DNA to the linear form. cellulose (Wako Pure Chem. Co.), [r-"P]-ATP One unit of the enzyme activity was defined as (New England Nuclear), [6-8H]-thymidine (Radio- the amount of the enzyme which converts 0.1 fig chemical Centre, Amersham), Ficoll (Pharmacia), of single-stranded circular fl DNA to the linear bovine serum albumin (Schwartz), ovalbumin form under the standard reaction conditions with (Schwartz), and myoglobin (Schwartz) were 0.4 fig of single-stranded circular f 1 DNA as a substrate, (b) The endonuclease activity for purchased from the indicated sources. DNA and RNA—»H-Labelled phage f 1 DNA (7,100 cpm/^g) was prepared as described by a b Marvin and Schaller (19). »H-Labelled fl doublestranded superhelical DNA (replicative form 1 DNA) was prepared as described by Model and Zinder (20). The final step of the purification was ethidium bromide/CsCl density gradient centrifugation. The specific radioactivity was 33,000 cpm//ig. Cold and 3H-labelled SPPl DNA circular were prepared as described by Riva et al. (21). S The specific radioactivity of the H-Iabelled SPPl linear DNA was 18,000 cpm//ig. Ribosomal RNA of B. subtilis was the kind gift of Dr. H. Takahashi. Assays of Endonuclease Activity—(a) The endonuclease activity for single-stranded DNA was assayed by measuring the conversion of singlestranded circular DNA of phage fl to the linear Fig. 1. Dose-response relation for endonuclease MIL Various amounts of enzyme diluted to about 1,000 form. This method is based on our finding that units/ml (Fraction V) were added to 25 ft] of the standsingle-stranded circular DNA and whole linear ard reaction mixture. Assays were conducted as deDNA can easily be separated by electrophoresis on scribed in " MATERIALS AND METHODS." Slots 1.6% agarose gel. The details of the method are a through g are the digestion products with 0, 0.5, 1, described in a separate paper (22). The standard 1.5, 2, 4, and 6 ft\ of Mil, respectively.
SINGLE-STRANDED DNA-SPECIFIC ENDONUCLEASE OF B. subtilis
0
1 2
4
S
ENDONUCLEASE M I K J J I )
Fig. 2. Dose-response curve for endonuclease MEL The radioactivity of each top-band (single-stranded circular DNA) of Fig. 1 was measured as described in " MATERIALS AND METHODS." The decreases in radioactivity of circular DNA in the reaction mixture are shown. double-stranded superhelical DNA was determined by measuring the conversion of 'H-labelled doublestranded superhelical DNA of f 1 to open-circular and linear DNA's by neutral and alkaline sucrose gradient sedimentations and by agarose gel electrophoresis. As these products form discrete bands on 0.7% agarose gel electrophoresis, the radioactivity of each DNA band could be measured. The reaction was carried out in the standard reaction mixture supplemented with NaQ to give a final concentration of 120 mM. (c) The nuclease activity for double-stranded linear DNA was assayed with SPP1 DNA. The DNA samples were analyzed qualitatively by 0.7% agarose gel electrophoresis and quantitatively by alkaline sucrose gradient sedimentation.
the presence of 0.5 /ig/ml of ethidium bromide. The 1.6% gel was stained after electrophoresis. DNA was visualized under ultraviolet light and photographed with Tri-X film (Kodak). To determine the radioactivity of each DNA band, the band was cut out from the gel plate and the agarose was hydrolyzed with ca. 0.1 N HC1. Sucrose Gradient Sedimentation—Neutral and alkaline sucrose gradient sedimentations of digestion products of f 1 DNA were done at 4°C in the Hitachi RPS65T rotor at 50,000 rpm. Neutral 5-20% sucrose gradients (4.7 ml) containing 0.9 M NaCl, 10 mM Tris-HCl, pH 8.0, and 5 mM EDTA were centrifuged for 180 min (single-stranded circular DNA) or for 240 min (double-stranded superhelical DNA), and alkaline sucrose gradients containing 0.25 N NaOH, 0.7 M NaCl, and 5 mM EDTA were centrifuged for 120 min (doublestranded superhelical DNA). Alkaline sucrose gradient sedimentation of digestion products of SPP1 DNA was carried out in the Hitachi RPS40T2 rotor at 38,000 rpm for 180 min. Two-drop fractions were collected directly onto GB100R glassfilters(Toyo Roshi Co. Ltd.) from the bottom of the polyethylene tube. The radioactivity was measured as described previously. The molecular weight of DNA was estimated according to the method of Abelson and Thomas (23).
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400
629
Glycerol Gradient Sedimentation—Sedimentation of endonuclease Mil was done at 4°C in the Hitachi RPS65T rotor at 44,000 rpm for 14 h. Gradients of 10-30% glycerol containing 20 mM Tris-HCl, pH 7.5, and 2 mM 2-mercaptoethanol were used. Fraction V of the enzyme was dialyzed against buffer B containing 7% glycerol prior to centrifugation. After centrifugation, two-drop fractions were collected from the bottom and the Agarose Gel Electrophoresis—The apparatus endonuclease activity was assayed by the standard for agarose slab gel electrophoresis was the vertical procedure. model of Studier (24) with minor modifications. Polynucleotide Kinase Treatment—PhosphoAgarose gels were prepared by dissolving agarose rylation of the endonuclease-digested DNA by (0.7% or 1.6% w/v, Sigma) in TAE buffer (40 mM T4 polynucleotide kinase followed the method Tris-acetate, 5 mM sodium acetate, 1 mM EDTA, described by Panet et al. (18). The reaction pH 7.8) and poured into a mold between two glass mixture (100 p\) contained 50 mM Tris-HCl (pH plates (140x120x4 mm). Samples were placed 8.0), 10 mM MgCl,, 5 mM 2-mercaptoethanol, in each well on the slab gel after being mixed with 2 mM KH,PO4, 2 mM spermine, 4 ftg of DNA, one-fifth volume of SEBP solution- Electro- 100 (iM [T-"P]-ATP (114,000 cpm), and 0.2 unit phoresis was carried out at 110 V at room tem- of T4 polynucleotide kinase (Ultrogel AcA34 perature for 2-3 h in TAE buffer. In the case filtrate fraction). The reaction mixture was incuof the 0.7 % gel, electrophoresis was carried out in bated at 37°C for 30 min. Vol. 86, No. 3, 1979
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K. MATSUMOTO, T. ANDO, H. SATTO, and Y. DCEDA
RESULTS
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Purification of Endonuclease Mil—Preparation of extract: TI strain of B. subtilis Marburg was grown in 6 liters of tryptone yeast extract medium and collected at the early stationary phase by centrifugation. The cells (20 g) were suspended in 70 ml of Buffer B (20 HIM Tris-HCl pH 7.5, 2 rrun 2-mercaptoethanol, 10% glycerol) containing 10 mM 2-mercaptoethanol and were treated with 50 /ig/ml of lysozyme at 37°C for 30 min. The protoplasts produced were subjected to sonic oscillation (4 times for 30 s each at a setting of 4.5) with an ultrasonic disruptor (UR-200P, Tomy Seiko Co.). 30 40 50 60 The cell debris was removed by centrifugation in FRACTION WJMBER the Hitachi RP30 rotor at 28,000 rpm for 90 min. Fig. 3. DEAE-cellulose chromatography of Fraction The supernatant (69 ml) was collected and desig- II. Endonuclease activity towards single-stranded cirnated as Fraction I. cular DNA was assayed in the standard reaction Ammonium sulfate precipitation: Powdered mixture. Endonuclease activity (•); protein concenammonium sulfate (21.3 g) was added to fraction tration measured as absorbance at 280 nm (O); molarity ). I with stirring. After further stirring for 30 min, of KCl ( the precipitate was removed by centrifugation. Powdered ammonium sulfate (19.8 g) was added also contained activity towards single-stranded slowly to the supernatant (79 ml) with stirring for DNA and towards double-stranded DNA in the 30 min. The precipitate was collected by centrifu- presence of CaClj or MgCla (data not shown). gation and dissolved in Buffer B (Fraction n, The fraction at 0.17-0.19 M KCl (tube Nos. 45-47) 50 ml). was pooled and concentrated to 11 ml using Ficoll DEAE-cellulose chromatography: Fraction II (Fraction HI). was dialyzed against Buffer B and applied to a Gel filtration: The concentrated Fraction i n DEAE-cellulose column (2.5x11 cm) previously was loaded on an Ultrogel AcA 34 column (2.5 x equilibrated with Buffer B. The column was 44 cm) previously equilibrated with Buffer B washed with the buffer and eluted with a linear (supplemented with 0.1 M KCl) and washed with a gradient of 0 to 0.3 M KC1 in Buffer B (300 ml) small volume of the buffer. Elution was carried at the flow rate of 17ml/h; fractions of 7 ml were out with the same buffer at the flow rate of 15 ml/h collected. Figure 3 shows the distribution pattern and fractions of 4.9 ml were collected. Figure of endonuclease activity for single-stranded DNA. 4 shows the distribution pattern of the endonuclease The nuclease activity for double-stranded DNA activity. The fractions exhibiting high activity was also assayed qualitatively with double-stranded towards single-stranded DNA but no activity on linear DNA of SPP1 under the same reaction con- double-stranded DNA (tube Nos. 29-32) were ditions by means of agarose gel electrophoresis. pooled (Fraction IV, 19.6 ml). Among the three peaks of activity towards singleThe Second DEAE-cellulose chromatography: stranded DNA eluted from the column, the fraction Fraction IV was dialyzed against Buffer B and at 0.17-0.19 M KC1 exhibited no activity on double- loaded onto a DEAE-cellulose column (2.5 x 3 cm) stranded DNA. However, the fraction eluted at previously equilibrated with Buffer B (supplemented 0.1-0.14 M KC1 exhibited high activity towards with 0.1 M KCl). The column was washed with double-stranded DNA in the presence of MnCli the buffer and the enzyme was eluted with 300 ml or CaClj. The fraction at 0.2-0.25 M KC1 ex- of a linear gradient of 0.1 to 0.3 M KCl in Buffer hibited enhanced activity for double-stranded DNA B. Theflowrate was 30 ml/h, and 3.5 ml fractions in the presence of ATP. The unadsorbed fraction were collected. The peak of the enzyme activity
SINGLE-STRANDED DNA-SPECIFIC ENDONUCLEASE OF B. subtilis
20
30
40
SO
FRACTION NUMBER
Fig. 4. Gel filtration of Fraction III on Ultrogel AcA34. Endonuclease activity towards single-stranded circular DNA was assayed in the standard reaction mixture. Endonuclease activity ( • ) ; protein concentration measured as absorbance at 280 nm (O).
30 ml of a 0.05 to 1.0 M KC1 gradient. The flow rate was 4.5 ml/h, and 1 ml fractions were collected. The fractions which exhibited high activity (eluted at 0.3-0.45 M KC1) were pooled (Fraction VI, 5 ml). DEAE-celluIose concentration: Four ml of Fraction VI was dialyzed against Buffer B and adsorbed onto a DEAE-cellulose column (0.5 x 1.2 cm) previously equilibrated with Buffer B (containing 20% glycerol). The enzyme was eluted with the same buffer containing 0.25 M K G . Fractions of 0.6 ml were collected and the most active fraction was used in the present experiments (Fraction VII, 0.6 ml). These fractions (V, VL, and VII) were supplemented with glycerol (final concentration, 50%) and stocked at - 2 0 ° C . The purification procedure is summarized in Table I. The enzyme was purified more than 320 times on a specific activity basis. Properties of Endonuclease Mil—Stability: The enzyme was labile after the second DEAEcellulose chromatography. Addition of ethylene glycol or glycerol stabilized the enzyme activity. The enzyme retained more than 90 % of its original activity in Buffer B containing 50% glycerol after storage for five months at —20°C. Optimal pH for action: The activity versus pH relation was assayed in a reaction mixture which contained 5 mM MnClj. The optimal p H was found to be between pH 8 and 9,40% and 10% of the optimal activity being observed at pH 7 and pH 10, respectively.
TABLE I. Purification of endonuclease Mil. The activity was assayed under the standard reaction conditions. Protein concentration was measured according to the method described by Lowry et al. (25). Fraction I. Crude extract II. Ammonium sulfate III. IV. V. VI. VII.
DEAE-cellulose I AcA 34 filtration DEAE-cellulose 11 DNA-cellulose* DEAE-cellulose concentration
Total protein (mg)
Total activity (units XlO"')
Specific activity (units xlO-'/ m 8 protein)
4,026 1,410 338 32.9 3.19 0.228 0.16
9,662 8,800 3,360 1,848 504 175 123
2.4 6.2 9.2 56.2 158 768 770
* Only a portion of Fraction V was used for further purification by denatured DNA-cellulose chromatography; the data presented were recalculated accordingly. Vol. 86, No. 3, 1979
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appeared at 0.17-0.19 M KC1. At this purification step, the fraction was apparently free from nuclease activity towards double-stranded DNA, even on prolonged incubation. The peak fractions were pooled (Fraction V, 14 ml). Denatured DNA-cellulose chromatography: Three ml of Fraction V was dialyzed extensively against Buffer C (20 mM Tris-HCl, pH 7.5, 2 rriM 2-mercaptoethanol, 1 mM EDTA, 50 mM KC1, and 10% glycerol) and loaded onto a denatured DNAcellulose column (0.8x2.8 cm) equilibrated with Buffer C previously. The column was washed with Buffer C and the enzyme was eluted with
631
632
K. MATSUMOTO, T. ANDO, H. SA1TO, and Y. IKEDA 1 mM and 25 mM potassium phosphate, respectively. Effect of RNA: When the enzyme was assayed in the presence of 2.1 or 4.2 fig per 25 pi of B. subtilis ribosomal RNA, the activity was inhibited by 35 % or 65 %, respectively. Absence of exonuclease activity: Singlestranded circular f 1 DNA was treated with 5, 10, and 20 units of the enzyme (Fraction V) and the digestion products were analyzed by sucrose gradient sedimentation. The absence of exonuclease activity was confirmed by the absence of nonsedimentable material (data not shown; refer to Figs. 7 and 10). Absence of phosphatase activity: The enzyme had no phosphomonoesterase activity, as indicated by the negligible formation of p-nitrophenol in the standard reaction mixture and in 1 M Tris-HCl buffer (pH 8.0) containing 1 mM p-nitrophenyl phosphate and 200 units of the enzyme (fraction V). Molecular weight: The size of the endonuclease was estimated by the gel filtration method according to Andrews (26). The estimated molecular size of Mil protein was 57,000 daltons as shown in Fig. 6. Glycerol gradient centrifugation of the enzyme was also carried out, and a Svedberg value of 4.2 was obtained by comparison with bovine serum albumin. Substrate Specificity of the Enzyme—Absence of action on double-stranded linear DNA: Throughout the purification procedure the endo-
20
T4 potynucleotid* klnase
"10 x 8
Ineseum albunin
I:
Mil
ovalbumln
myoglobln 1.2 2
5
10
20
50
( mM ) Fig. 5. Effects of divalent cations on the activity. MnCl, (5 mM) in the standard reaction mixture was replaced by the indicated concentrations of CaCl- (•), MnCl, (O), and MgCl, (•). Each activity was expressed as a percentage of the activity obtained with 5 mM MnCl,. CATION
1-4
16 Vtf Vo
18
2-0
Fig. 6. Estimation of molecular size by gel filtration. MH was loaded on an Ultrogel AcA34 column (1 x 52 cm) and eluted with Buffer B containing 0.1 M NaCl. The following proteins were used as standards: T4 polynucleotide kinase, M.W. 140,000 (18); bovine serum albumin, M.W. 67,000; ovalbumin, M.W. 45,000; myoglobin, M.W. 17,800. /. Biochenu
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Effect of divalent cations on the activity: Figure 5 shows plots of relative activity versus divalent cation concentration. CaCl, was the most effective (at 2-50 mM). The optimal concentrations for MnCl, and MgQ, were 2-5 mM and 5-20 mM, respectively. The activity was quite low in the absence of these cations. High concentrations of CaClt, although this was the most effective cation, often prevent the penetration of single-stranded DNA into 1.6% agarose gel in the presence of high concentrations of protein. Therefore, MnClt was used for the standard assay system. Effect of NaCl on the activity: The activity was not inhibited by the addition of 120 mM or 200 mM NaCl under the standard reaction conditions. Even at 500 mM NaCl, about 70% of the activity was still retained. Effect of ATP on the activity: The activity was not affected at all in the presence of 25 or 500 fJM ATP in reaction mixtures in which MnCl, was replaced by MgCla. Double-stranded linear DNA of SPPl was not hydrolyzed by endonuclease Mil (up to 20 units, incubation for 60 min) in the same reaction mixtures when they were tested by 0.7 % agarose gel electrophoresis. Effect of KtHPOt on the activity: Potassium phosphate inhibited the activity strongly. Inhibition reached 80% and 95% in the presence of
SINGLE-STRANDED DNA-SPECIFIC ENDONUCLEASE OF B. subtilis
calculated to be only 0.4 per double-stranded molecule. This result indicates that the endonuclease is highly specific for single-stranded DNA. Action on double-stranded superhelical DNA:
The enzyme (up to 4 units per 25 fi\ for 20 min) did not cleave double-stranded superhelical DNA of fl, as determined by 0.7% agarose gel electrophoresis (data not shown). As it has been reported that double-stranded superhelical DNA is cleaved by large amounts of nuclease SI (27, 28), a similar experiment was performed with high doses of ME using 'H-labelled double-stranded superhelical DNA of fl. To compare the action of MTJ (at high dose) on double-stranded superhelical DNA with that on single-stranded DNA, the time course of cleavage of single-stranded circular fl DNA with 18.2 units of Mil per 25 /il was tested. Within 0.75 min, 37% of the singlestranded circular DNA was cleaved. After two min, 85 % was cleaved, and after 20 min 98 % was cleaved to small fragments (data not shown). Figure 8 shows the cleavage pattern of doublestranded superhelical DNA by 18.2 units per
a b c d e f
oc L DSS
10 20 30 FRACTION (UMBER
Fig. 7. Alkaline sucrose gradient sedimentation of double-stranded linear DNA treated with ME. The standard reaction mixture (150 /il) containing 2.4 fig of s H-labelled double-stranded linear DNA of phage SPP1 (18,000 cpm//ig) was treated with 109 units of Mil. NaCl was added to make a final concentration of 120 IDM. At 0 min (A), after 60 min (B) and after 120 min ( Q of incubation, 50 /il samples were withdrawn, diluted into 150 /il of 5 HIM EDTA solution at 0°C and subjected to sedimentation analysis. Vol. 86, No. 3, 1979
Fig. 8. Time course of cleavage of double-stranded superhelical DNA with MIT. The standard reaction mixture (175 /il) containing 2.8 fig of sH-labelled doublestranded superhelical DNA of fl (33,000 cpm//ig) containing NaCl to give a final concentration of 120 HIM was treated with 127 units of ME. At intervals during incubation at 37°C, 25 fi\ samples were withdrawn and subjected to 0.7% agarose gel electrophoresis. Top, middle, and bottom bands correspond to opencircular, linear, and double-stranded superhelical DNA, respectively. Slots a through / are the digestion products incubated for 0, 10, 20, 40, 60, and 120 min, respectively.
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nuclease activity was assayed in two ways, i.e., with single-stranded circular fl DNA and with double-stranded linear DNA of SPP1 as substrates. As the latter assay was qualitative, the data are not shown. In order to confirm the specificity for single-stranded DNA, double-stranded DNA of SSP1 was treated with higher dose of Mil for longer incubation time and subjected to alkaline sucrose gradient sedimentation analysis (Fig. 7). The molecular size of the DNA treated with 18.2 units of Mil per 25 fi\ for 60min did not change, though after 120 min the molecular size became slightly smaller. The molecular size of the DNA treated for 120 min was estimated to be 10.4 x 10* daltons according to the equation of Abelson and Thomas (23), assuming the molecular size of alkali-denatured SPP1 DNA to be 12.5 x 10* daltons (21). The possible number of nicks introduced after incubation for 120 min was
633
634
K. MATSUMOTO, T. ANDO, H. SA1TO, and Y. DCEDA trations of NaCl, while the activity towards singlestranded circular DNA was not inhibited. Marked inhibition of the activity on double-stranded DNA by high concentrations of salts is a common feature of nucleases specific for single-stranded DNA (27-31). These results suggest that ME nicks either strand of double-stranded superhelical DNA at a weakly hydrogen-bonded region and then nicks the remaining strand gradually. Identification of termini of cleaved DNA: The digestion product of single-stranded circular fl DNA with Mil was tested for 5'-termini which would accept 31P from [;--"P]ATP in the presence of T4 polynucleotide kinase, using the method described by Panet et al. (18). Digestion products 2000
cpm
a
