JOURNAL OF BACTERIOLOGY, Mar. 1979, p. 1299-1307 0021-9193/79/03-1299/09$02.00/0
Vol. 137, No. 3
NgoII, a Restriction Endonuclease from Neisseria gonorrhoeae DAVID J. CLANTON,t W. STUART RIGGSBY, AND ROBERT V. MILLER* Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37916 Received for publication 23 December 1978
EndoR NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage A deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2"; maximal enzyme activity was observed at 1 mM Mg2e. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation. We recently reported (5) the discovery of a tained from Bethesda Research Laboratories, Inc. GC restriction endonuclease, NgoII, from clinical Agar Base was supplied by Difco, and IsoVitaleX was isolates of Neisseria gonorrhoeae. This enzyme obtained from BBL. Aldolase, ovalbumin, chymotrypwas found in four of seven isolates surveyed and sinogen A, and RNase A were obtained from WorthBiochemical Corp. Cytochrome c, lysozyme, had several properties of a class II restriction ington streptomycin sulfate, bovine serum albumin, and Sendonuclease. adenosyl-L-methionine were obtained from Sigma Class II endonucleases require only Mg2" as a Chemical Co. Phosphocellulose (Pll) was obtained cofactor for activity and will cleave unmodified from Whatman Ltd. Hydroxyapatite (Hypatite C) was DNA at a specific site within or at the end of obtained from Clarkson Chemical Co., Inc., and the DNA sequence which serves as the recogni- DEAE-cellulose was Cellex-D purchased from Biotion site for the enzyme (19). Because the cleav- Rad Laboratories. Agarose was SeaKem (ME) obage of DNA always occurs at the same site, the tained from Marine Colloids, Inc. Acrylamide and fragments produced from a particular DNA sub- other electrophoresis supplies were from Bio-Rad LabSephadex G-150 (superfine grade) and Blue strate will always be the same. Class II enzymes oratories. Dextran 2000 were purchased from Pharmacia Fine are distinguished from class I restriction endo- Chemicals. Sodium dodecyl sulfate (SDS) and all nucleases by the fact that, while class I restric- other reagents were obtained from Fisher Scientific tion endonucleases recognize specific nucleotide Co. sequences on unmodified DNA, they cleave the Bacterial strains and culture media. A protoDNA at a random point outside of the recogni- trophic strain, MUTK1, of N. gonorrhoeae was used tion sequence (11, 15). They require ATP, Mg2+, throughout this study (5). It was grown in gonococcal and S-adenosyl-L-methionine as cofactors for ac- biphasic growth medium, as described by Sparling (22). This medium was inoculated from a culture tivity (8, 10). on plates containing GC Agar Base This report continues our studies on EndoR- grown overnight with 1% IsoVitaleX. NgoII. It presents a purification scheme for the supplemented Fractionation steps used in enzyme purificaenzyme and defines several of the enzyme's tion. (i) Preparation of cell extracts. MUTK1 was physical and biochemical properties. These inoculated and grown in 3 liters of gonococcal biphasic properties confirm that NgoII is a class II re- growth medium for 12 to 14 h at 37°C. Cells were striction endonuclease. harvested by centrifugation, resuspended in 15 ml of 0.14 M NaCl, and incubated at 37°C for 10 min to MATERIALS AND METHODS deplete intracellular ATP. Cells were then reharvested Materials. Restriction endonucleases HaeIII and by centrifugation, and lysed by a modification of the EcoRI were obtained from New England Biolabs. Sim- method described by Miller and Clark (14). Cells were ian virus 40 DNA and phage lambda DNA were ob- suspended in a 10% sucrose solution prepared in 0.5 M Tris-hydrochloride at a concentration of 0.5 g/ml, t Present address: Department of Biocheniistry, George- frozen in an acetone-dry ice bath, and thawed at room town University School of Medicine, Washington, DC 20007. temperature. The volume was measured, and 0.1 ml of 1299
1300
CLANTON, RIGGSBY, AND MILLER
lysozyme solution (2 mg/ml) and 0.1 ml of 1 M NaCl were added for each milliliter of cell suspension. The mixture was incubated on ice for 60 to 75 min. All subsequent operations were carried out at 4°C. Cell debris was removed by centrifugation for 20 min at 35,000 x g, and the supernatant fluid (fraction I) was decanted and stored at -20°C until further treated. (ii) Streptomycin sulfate precipitation. Fraction I was treated by slow addition of a 10% solution of streptomycin sulfate with constant stirring to a final concentration of 1.7%. The mixture was stirred for an additional 20 min, and the precipitated DNA was removed by centrifugation for 20 min at 27,000 x g. The precipitate was discarded, and the supernatant fluid (fraction II) was further purified. (iii) Ammonium sulfate precipitation. Ammonium sulfate was added slowly to fraction II to a final concentration of 70% (wt/vol) and stirred for 20 min. The precipitate was recovered by centrifugation at 35,000 x g for 20 min and redissolved in buffer A (0.01 M potassium phosphate buffer, pH 7.0, 6 mM 2-mercaptoethanol, and 1 mM EDTA, prepared in a 10% glycerol solution) to which NaCl had been added to a final concentration of 0.02 M. This solution was then dialyzed for at least 4 h against buffer A containing 0.002 M NaCl. During dialysis, a precipitate sometimes formed that had no detectable endonuclease activity as assayed by the method described below. This precipitate was removed by centrifugation at 12,000 x g for 10 min. The supernatant fluid (fraction III) was assayed for endonuclease activity and was used for further purification. (iv) Phosphoceliulose chromatography. Fraction III was layered directly onto a phosphocellulose P11 column (0.9 cm by 20 cm) equilibrated with buffer A containing 0.02 M NaCl and was washed with 10 ml of buffer A. The column was eluted with a 200-ml gradient of 0.02 to 1.0 M NaCl prepared in buffer A. Fractions (3 ml) were collected, and even-numbered fractions were assayed for endonuclease activity by the method described below. Those fractions that displayed activity were pooled (fraction IV) and stored at -20°C until further treatment. (v) Hydroxyapatite concentration. Fraction IV was layered onto a hydroxyapatite column (0.5 by 5 cm) equilibrated in buffer A containing 0.1 M NaCl. The column was washed with 2 ml of buffer A, and the endonuclease activity was eluted with buffer B (0.7 M potassium phosphate buffer, pH 7.0, 0.1 M NaCl, 1 mM EDTA, and 6 mM 2-mercaptoethanol prepared in a solution of 10% glycerol). Fractions (1 ml) were collected, and each was dialyzed separately in buffer A containing 0.02 M NaCl and assayed. Fractions that contained endonuclease activity were pooled (fraction V) and stored at -20°C until further treated. (vi) DEAE-cellulose chromatography. DEAEcellulose was equilibrated with buffer A (prepared in a solution of 50% glycerol), and a column 0.5 by 5.0 cm was poured. Fraction V was layered onto the column, which was then washed with 5 ml of buffer A containing 0.02 M NaCl and eluted with a 0.02 to 1.0 M NaCl gradient. Each fraction (0.5 ml) was assayed for endonuclease activity, and those fractions that contained activity were pooled (fraction VI) and stored at -20°C.
J. BACTERIOL.
Assay for endonuclease activity. The assay for endonuclease activity is a modification of the agarose gel assay first described by Sharp et al. (21). The standard reaction mixture contained 0.5 ug of substrate DNA, 0.1 M Tris-hydrochloride (pH 7.5), 0.02 M NaCl, 5 mM MgCl2, 2 ,ug of bovine serum albumin, and 5 ,l of sample endonuclease. The total reaction volume was 20 pd. Reaction mixtures were usually incubated at 37°C for 1 h. The reaction was initiated by the addition of the sample to be assayed and was terminated by the addition of 5 pl of a solution containing 5% SDS, 25% glycerol, and 0.025% bromophenol blue. The amount of sample added to the reaction mixture, the incubation time, or the DNA substrate species was occasionally varied as noted in the Results. When it was necessary to quantitate the number of units of enzyme activity in a sample, the endonuclease sample was serially diluted in twofold steps, and 5 Ml of each dilution was added to separate reaction mixtures containing 1 Mug of substrate DNA. After 1 h of incubation at 37°C, the reaction was terminated as described above, and the samples were placed on agarose gels for electrophoresis (see below). The highest dilution of sample that produced a limit digest was determined. We define 1 U of endonuclease as that amount of enzyme which produces a limit digest of 1 ,ug of A DNA in 1 h. After termination of the endonuclease reaction, samples were loaded onto a 1.4% agarose slab gel (20 by 20 by 0.3 cm) prepared in buffer C (89 mM Trizma base, 89 mM boric acid, and 0.25 mM EDTA, pH 7.4). Electrophoresis was carried out in buffer C at 150 V for 1 h or until the bromophenol blue neared the end of the gel. Migration was from cathode to anode. The gel was stained for 1 h in a solution of 0.5 tig of ethidium bromide per ml of buffer C and was photographed under UV irradiation (9). Assay for methylase activity. Endonuclease assay mixtures were prepared as described above, except that MgCl2 was not included and S-adenosyl-L-methionine was added at a final concentration of either 1 or 10IMM. Incubation of 1 ug of phage A DNA with 5 ul of endonuclease from fraction IV was allowed to proceed for 45 min in the presence of S-adenosyl-L-methionine without MgCl2. MgCl2 at a final concentration of 5 mM and an additional 5Ml of endonuclease were then added to cleave any unmodified DNA. After an additional 50 min, the assay mixtures were loaded onto 1.4% agarose slab gels and subjected to electrophoresis as described above. Protein determinations. Protein determinations were made by the method of Lowry et al. (12). Bovine serum albumin was used as a standard. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was performed to determine the purity of endonuclease after each fractionation step. Ten percent polyacrylamide gels (0.5 by 10 cm) were prepared in gel buffer (7.8 g of NaH2PO4. H20, 20.45 g of Na2HPO4, dissolved in 1 liter of deionized water, pH 7.2). Separate 50-Ml samples from each of fractions I-VI were added to 5 ul of tracking dye (0.125% bromophenol blue in 40% sucrose solution), and these mixtures were layered onto individual gels. Electrophoresis was carried out in gel buffer diluted 1:
IVOL. 137, 1979
RESTRICTION ENDONUCLEASE FROM N. GONORRHOEAE
1 with water until the dye neared the end of the gel. Migration was from cathode to anode. The gels were removed from the tubes and stained with Coomassie brilliant blue G-250 by the method of Reisner et al. (16). Molecular weight determinations. (i) Sephadex G-150 chromatography. Chromotography with Sephadex G-150 superfine was used to determine the molecular weight (1, 6) of the restriction endonuclease NgoII. A column 0.9 by 50 cm was poured and washed extensively with buffer A containing 0.1 M NaCl in 50% glycerol. The column was calibrated with the following proteins of known molecular weight: cytochrome c (12,700), chymotrypsinogen A (25,000), ovalbumin (45,000), and aldolase (158,000). The void volume was determined with Blue Dextran 2,000. The elution volume of the standard proteins was determined and the Kav was calculated (6). After calibration of the Sephadex G-150 column, 0.5 ml of the phosphocellulose fraction (fraction IV) was applied, and the protein was eluted with buffer A. Fractions (0.5 ml) were collected, and each was assayed for endonuclease activity. The elution volume was determined, and the molecular weight of EndoR.NgoII was estimated by extrapolation of a straight-line graph produced by plotting the log molecular weight of the standard proteins as a function of their respective Kay's. (ii) SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed by methods previously described (13, 24) to determine the molecular weight of the denatured endonuclease. Ten percent polyacrylamide gels (0.4 by 10 cm) were prepared in gel buffer (7.8 g of NaH2PO4. H20, 20.45 g of Na2HPO4, and 2 g of SDS per liter). Protein samples were made 1% with respect to SDS and placed in a boiling-water bath for 1 min. Five microliters of a 0.125% bromophenol blue solution prepared in 40% sucrose was added to each protein, and the samples were layered onto the gels. Electrophoresis was carried out in gel buffer diluted 1:1 with water for 4 h at 8 mA/gel. Migration was from cathode to anode. After electrophoresis, the gels were removed from the tubes, and the lengths of each gel were measured. The gels were then stained for protein with 0.025% Coomassie brilliant blue dissolved in a solution of 50% methanol and 7.5% acetic acid for 4 h and destained in a solution of 5% methanol and 7.5% acetic acid overnight. The lengths of the gels after destaining and the positions of the blue protein bands were recorded. The standard proteins used for the Sephadex G-150 chromatography were also used for the calibration of the gels except that RNase A (molecular weight, 13,700) replaced cytochrome c.
1301
RESULTS Purification of EndoR.NgoII. NgoII was purified from N. gonorrhoeae strain MUTK1. After lysis, the cell extract was examined for endonuclease activity; however, the presence of other exo- and endonucleases made a determination of restriction endonuclease activity impossible at this point. After treatment with streptomycin sulfate, the lysate was precipitated with 70% (wt/vol) (NH4)2SO4. Approximately 40% of the protein in the lysate was recovered by (NH4)2SO4 precipitation. An examination of this fraction revealed that the restriction endonuclease activity was precipitated by the addition of this concentration of (NH4)2SO4 (Table 1).
The (NH4)2SO4 fraction (fraction III) was layered onto a phosphocellulose column and eluted with a 0.02 to 1.0 M NaCl gradient. The restriction endonuclease activity eluted with the peak of activity at approximately 0.2 M NaCl (Fig. 1 and Table 1). A second peak of activity, which we believe to be NgoI (19), was separated from NgoII by elution on phosphocellulose (Fig. 1). The NgoII activity which eluted from the phosphocellulose column was not stable at -20°C without the addition of 10% glycerol in the elution buffer. Consequently, all subsequent operations were performed in 10% glycerol; under these conditions the activity was retained for at least 2 months at -20°C. The pooled fractions from the phosphocellulose column (fraction IV) were applied to a hydroxyapatite column and eluted with buffer B. The endonuclease activity was removed from the hydroxyapatite as a broad peak (fraction V). At this stage, the specific activity of the endonuclease was 1.56 U/,ug of protein, and the percent recovery as compared to the (NH4)2SO4 fraction was 29% (Table 1). Fraction V was free from any detectable contaminating nonspecific nucleases. This was shown by extensive incubation (4 h) of the pooled fractions with phage A DNA. The sharpness of the bands produced after gel electrophoresis was not affected by this extensive incubation. Fraction V was further purified by DEAE-
TABLE 1. Purification of EndoR * NgoII Fractiona
Vol (ml)
Total protein (mg)
Total U
Sp act (U/ Purificamg of protein)
tion
Percent re-
covery
100 345 1.0 111.8 III. (NH4)2SO4, precipitate .29 38,580 47 2.7 938 19.2 IV. Phosphocellulose chromatography 13.5 18,000 29 4.5 7.3 V. Hydroxyapatite concentration .12.2 11,380 1,556 38.8 12 0.34 5.7 VI. DEAE-cellulose chromatography 4,560 13,411 a Quantitative determinations of NgoII activity could not be made in fractions I and II. See text for
explanation.
1302
CLANTON, RIGGSBY, AND MILLER
J. BACTERIOL.
FIG. 1. Agarose gel electrophoresis patterns ofphage A DNA digested with fractions from phosphocellulose chromatography of lysates of strain MUTK1. Incubation conditions were as described in the text. After incubations, the digested DNA was subjected to gel electrophoresis in a 1.4% agarose gel and stained with ethidium bromide. NaCl concentration of the eluting buffer increases from left to right. EndoR. NgoII eluted at 0.18 M NaCl (wells 8-10). The fractions corresponding to these wells were pooled for further purification. An activity similar to that reported for EndoR.NgoI (19) eluted earlier (0.09 M NaCl, well 5).
TABLE 2. Effect of temperature on EndoR. NgoII (fraction V)
Purity of the restriction endonuclease. The purity of the restriction endonuclease was Percentage of maximal activitya determined by polyacrylamide gel electrophoresis. Amounts of 50 pl of each of fractions I-VI Temp (°C) Enzyme activwere layered onto 10% polyacrylamide gels, and, Enzyme stability' ityh after electrophoresis, the gels were stained for 4 0 NDd protein and photographed. The DEAE-cellulose 25 12 ND fraction (fraction VI) contained a single band of 35 50 100 protein (Fig. 2A). A parallel, unstained gel was 45 50 100 sliced into 2-mm segments, and each segment 55 100 6 was crushed in 0.1 ml of buffer A in separate 65
Vol. 137, No. 3
NgoII, a Restriction Endonuclease from Neisseria gonorrhoeae DAVID J. CLANTON,t W. STUART RIGGSBY, AND ROBERT V. MILLER* Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37916 Received for publication 23 December 1978
EndoR NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage A deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2"; maximal enzyme activity was observed at 1 mM Mg2e. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation. We recently reported (5) the discovery of a tained from Bethesda Research Laboratories, Inc. GC restriction endonuclease, NgoII, from clinical Agar Base was supplied by Difco, and IsoVitaleX was isolates of Neisseria gonorrhoeae. This enzyme obtained from BBL. Aldolase, ovalbumin, chymotrypwas found in four of seven isolates surveyed and sinogen A, and RNase A were obtained from WorthBiochemical Corp. Cytochrome c, lysozyme, had several properties of a class II restriction ington streptomycin sulfate, bovine serum albumin, and Sendonuclease. adenosyl-L-methionine were obtained from Sigma Class II endonucleases require only Mg2" as a Chemical Co. Phosphocellulose (Pll) was obtained cofactor for activity and will cleave unmodified from Whatman Ltd. Hydroxyapatite (Hypatite C) was DNA at a specific site within or at the end of obtained from Clarkson Chemical Co., Inc., and the DNA sequence which serves as the recogni- DEAE-cellulose was Cellex-D purchased from Biotion site for the enzyme (19). Because the cleav- Rad Laboratories. Agarose was SeaKem (ME) obage of DNA always occurs at the same site, the tained from Marine Colloids, Inc. Acrylamide and fragments produced from a particular DNA sub- other electrophoresis supplies were from Bio-Rad LabSephadex G-150 (superfine grade) and Blue strate will always be the same. Class II enzymes oratories. Dextran 2000 were purchased from Pharmacia Fine are distinguished from class I restriction endo- Chemicals. Sodium dodecyl sulfate (SDS) and all nucleases by the fact that, while class I restric- other reagents were obtained from Fisher Scientific tion endonucleases recognize specific nucleotide Co. sequences on unmodified DNA, they cleave the Bacterial strains and culture media. A protoDNA at a random point outside of the recogni- trophic strain, MUTK1, of N. gonorrhoeae was used tion sequence (11, 15). They require ATP, Mg2+, throughout this study (5). It was grown in gonococcal and S-adenosyl-L-methionine as cofactors for ac- biphasic growth medium, as described by Sparling (22). This medium was inoculated from a culture tivity (8, 10). on plates containing GC Agar Base This report continues our studies on EndoR- grown overnight with 1% IsoVitaleX. NgoII. It presents a purification scheme for the supplemented Fractionation steps used in enzyme purificaenzyme and defines several of the enzyme's tion. (i) Preparation of cell extracts. MUTK1 was physical and biochemical properties. These inoculated and grown in 3 liters of gonococcal biphasic properties confirm that NgoII is a class II re- growth medium for 12 to 14 h at 37°C. Cells were striction endonuclease. harvested by centrifugation, resuspended in 15 ml of 0.14 M NaCl, and incubated at 37°C for 10 min to MATERIALS AND METHODS deplete intracellular ATP. Cells were then reharvested Materials. Restriction endonucleases HaeIII and by centrifugation, and lysed by a modification of the EcoRI were obtained from New England Biolabs. Sim- method described by Miller and Clark (14). Cells were ian virus 40 DNA and phage lambda DNA were ob- suspended in a 10% sucrose solution prepared in 0.5 M Tris-hydrochloride at a concentration of 0.5 g/ml, t Present address: Department of Biocheniistry, George- frozen in an acetone-dry ice bath, and thawed at room town University School of Medicine, Washington, DC 20007. temperature. The volume was measured, and 0.1 ml of 1299
1300
CLANTON, RIGGSBY, AND MILLER
lysozyme solution (2 mg/ml) and 0.1 ml of 1 M NaCl were added for each milliliter of cell suspension. The mixture was incubated on ice for 60 to 75 min. All subsequent operations were carried out at 4°C. Cell debris was removed by centrifugation for 20 min at 35,000 x g, and the supernatant fluid (fraction I) was decanted and stored at -20°C until further treated. (ii) Streptomycin sulfate precipitation. Fraction I was treated by slow addition of a 10% solution of streptomycin sulfate with constant stirring to a final concentration of 1.7%. The mixture was stirred for an additional 20 min, and the precipitated DNA was removed by centrifugation for 20 min at 27,000 x g. The precipitate was discarded, and the supernatant fluid (fraction II) was further purified. (iii) Ammonium sulfate precipitation. Ammonium sulfate was added slowly to fraction II to a final concentration of 70% (wt/vol) and stirred for 20 min. The precipitate was recovered by centrifugation at 35,000 x g for 20 min and redissolved in buffer A (0.01 M potassium phosphate buffer, pH 7.0, 6 mM 2-mercaptoethanol, and 1 mM EDTA, prepared in a 10% glycerol solution) to which NaCl had been added to a final concentration of 0.02 M. This solution was then dialyzed for at least 4 h against buffer A containing 0.002 M NaCl. During dialysis, a precipitate sometimes formed that had no detectable endonuclease activity as assayed by the method described below. This precipitate was removed by centrifugation at 12,000 x g for 10 min. The supernatant fluid (fraction III) was assayed for endonuclease activity and was used for further purification. (iv) Phosphoceliulose chromatography. Fraction III was layered directly onto a phosphocellulose P11 column (0.9 cm by 20 cm) equilibrated with buffer A containing 0.02 M NaCl and was washed with 10 ml of buffer A. The column was eluted with a 200-ml gradient of 0.02 to 1.0 M NaCl prepared in buffer A. Fractions (3 ml) were collected, and even-numbered fractions were assayed for endonuclease activity by the method described below. Those fractions that displayed activity were pooled (fraction IV) and stored at -20°C until further treatment. (v) Hydroxyapatite concentration. Fraction IV was layered onto a hydroxyapatite column (0.5 by 5 cm) equilibrated in buffer A containing 0.1 M NaCl. The column was washed with 2 ml of buffer A, and the endonuclease activity was eluted with buffer B (0.7 M potassium phosphate buffer, pH 7.0, 0.1 M NaCl, 1 mM EDTA, and 6 mM 2-mercaptoethanol prepared in a solution of 10% glycerol). Fractions (1 ml) were collected, and each was dialyzed separately in buffer A containing 0.02 M NaCl and assayed. Fractions that contained endonuclease activity were pooled (fraction V) and stored at -20°C until further treated. (vi) DEAE-cellulose chromatography. DEAEcellulose was equilibrated with buffer A (prepared in a solution of 50% glycerol), and a column 0.5 by 5.0 cm was poured. Fraction V was layered onto the column, which was then washed with 5 ml of buffer A containing 0.02 M NaCl and eluted with a 0.02 to 1.0 M NaCl gradient. Each fraction (0.5 ml) was assayed for endonuclease activity, and those fractions that contained activity were pooled (fraction VI) and stored at -20°C.
J. BACTERIOL.
Assay for endonuclease activity. The assay for endonuclease activity is a modification of the agarose gel assay first described by Sharp et al. (21). The standard reaction mixture contained 0.5 ug of substrate DNA, 0.1 M Tris-hydrochloride (pH 7.5), 0.02 M NaCl, 5 mM MgCl2, 2 ,ug of bovine serum albumin, and 5 ,l of sample endonuclease. The total reaction volume was 20 pd. Reaction mixtures were usually incubated at 37°C for 1 h. The reaction was initiated by the addition of the sample to be assayed and was terminated by the addition of 5 pl of a solution containing 5% SDS, 25% glycerol, and 0.025% bromophenol blue. The amount of sample added to the reaction mixture, the incubation time, or the DNA substrate species was occasionally varied as noted in the Results. When it was necessary to quantitate the number of units of enzyme activity in a sample, the endonuclease sample was serially diluted in twofold steps, and 5 Ml of each dilution was added to separate reaction mixtures containing 1 Mug of substrate DNA. After 1 h of incubation at 37°C, the reaction was terminated as described above, and the samples were placed on agarose gels for electrophoresis (see below). The highest dilution of sample that produced a limit digest was determined. We define 1 U of endonuclease as that amount of enzyme which produces a limit digest of 1 ,ug of A DNA in 1 h. After termination of the endonuclease reaction, samples were loaded onto a 1.4% agarose slab gel (20 by 20 by 0.3 cm) prepared in buffer C (89 mM Trizma base, 89 mM boric acid, and 0.25 mM EDTA, pH 7.4). Electrophoresis was carried out in buffer C at 150 V for 1 h or until the bromophenol blue neared the end of the gel. Migration was from cathode to anode. The gel was stained for 1 h in a solution of 0.5 tig of ethidium bromide per ml of buffer C and was photographed under UV irradiation (9). Assay for methylase activity. Endonuclease assay mixtures were prepared as described above, except that MgCl2 was not included and S-adenosyl-L-methionine was added at a final concentration of either 1 or 10IMM. Incubation of 1 ug of phage A DNA with 5 ul of endonuclease from fraction IV was allowed to proceed for 45 min in the presence of S-adenosyl-L-methionine without MgCl2. MgCl2 at a final concentration of 5 mM and an additional 5Ml of endonuclease were then added to cleave any unmodified DNA. After an additional 50 min, the assay mixtures were loaded onto 1.4% agarose slab gels and subjected to electrophoresis as described above. Protein determinations. Protein determinations were made by the method of Lowry et al. (12). Bovine serum albumin was used as a standard. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was performed to determine the purity of endonuclease after each fractionation step. Ten percent polyacrylamide gels (0.5 by 10 cm) were prepared in gel buffer (7.8 g of NaH2PO4. H20, 20.45 g of Na2HPO4, dissolved in 1 liter of deionized water, pH 7.2). Separate 50-Ml samples from each of fractions I-VI were added to 5 ul of tracking dye (0.125% bromophenol blue in 40% sucrose solution), and these mixtures were layered onto individual gels. Electrophoresis was carried out in gel buffer diluted 1:
IVOL. 137, 1979
RESTRICTION ENDONUCLEASE FROM N. GONORRHOEAE
1 with water until the dye neared the end of the gel. Migration was from cathode to anode. The gels were removed from the tubes and stained with Coomassie brilliant blue G-250 by the method of Reisner et al. (16). Molecular weight determinations. (i) Sephadex G-150 chromatography. Chromotography with Sephadex G-150 superfine was used to determine the molecular weight (1, 6) of the restriction endonuclease NgoII. A column 0.9 by 50 cm was poured and washed extensively with buffer A containing 0.1 M NaCl in 50% glycerol. The column was calibrated with the following proteins of known molecular weight: cytochrome c (12,700), chymotrypsinogen A (25,000), ovalbumin (45,000), and aldolase (158,000). The void volume was determined with Blue Dextran 2,000. The elution volume of the standard proteins was determined and the Kav was calculated (6). After calibration of the Sephadex G-150 column, 0.5 ml of the phosphocellulose fraction (fraction IV) was applied, and the protein was eluted with buffer A. Fractions (0.5 ml) were collected, and each was assayed for endonuclease activity. The elution volume was determined, and the molecular weight of EndoR.NgoII was estimated by extrapolation of a straight-line graph produced by plotting the log molecular weight of the standard proteins as a function of their respective Kay's. (ii) SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed by methods previously described (13, 24) to determine the molecular weight of the denatured endonuclease. Ten percent polyacrylamide gels (0.4 by 10 cm) were prepared in gel buffer (7.8 g of NaH2PO4. H20, 20.45 g of Na2HPO4, and 2 g of SDS per liter). Protein samples were made 1% with respect to SDS and placed in a boiling-water bath for 1 min. Five microliters of a 0.125% bromophenol blue solution prepared in 40% sucrose was added to each protein, and the samples were layered onto the gels. Electrophoresis was carried out in gel buffer diluted 1:1 with water for 4 h at 8 mA/gel. Migration was from cathode to anode. After electrophoresis, the gels were removed from the tubes, and the lengths of each gel were measured. The gels were then stained for protein with 0.025% Coomassie brilliant blue dissolved in a solution of 50% methanol and 7.5% acetic acid for 4 h and destained in a solution of 5% methanol and 7.5% acetic acid overnight. The lengths of the gels after destaining and the positions of the blue protein bands were recorded. The standard proteins used for the Sephadex G-150 chromatography were also used for the calibration of the gels except that RNase A (molecular weight, 13,700) replaced cytochrome c.
1301
RESULTS Purification of EndoR.NgoII. NgoII was purified from N. gonorrhoeae strain MUTK1. After lysis, the cell extract was examined for endonuclease activity; however, the presence of other exo- and endonucleases made a determination of restriction endonuclease activity impossible at this point. After treatment with streptomycin sulfate, the lysate was precipitated with 70% (wt/vol) (NH4)2SO4. Approximately 40% of the protein in the lysate was recovered by (NH4)2SO4 precipitation. An examination of this fraction revealed that the restriction endonuclease activity was precipitated by the addition of this concentration of (NH4)2SO4 (Table 1).
The (NH4)2SO4 fraction (fraction III) was layered onto a phosphocellulose column and eluted with a 0.02 to 1.0 M NaCl gradient. The restriction endonuclease activity eluted with the peak of activity at approximately 0.2 M NaCl (Fig. 1 and Table 1). A second peak of activity, which we believe to be NgoI (19), was separated from NgoII by elution on phosphocellulose (Fig. 1). The NgoII activity which eluted from the phosphocellulose column was not stable at -20°C without the addition of 10% glycerol in the elution buffer. Consequently, all subsequent operations were performed in 10% glycerol; under these conditions the activity was retained for at least 2 months at -20°C. The pooled fractions from the phosphocellulose column (fraction IV) were applied to a hydroxyapatite column and eluted with buffer B. The endonuclease activity was removed from the hydroxyapatite as a broad peak (fraction V). At this stage, the specific activity of the endonuclease was 1.56 U/,ug of protein, and the percent recovery as compared to the (NH4)2SO4 fraction was 29% (Table 1). Fraction V was free from any detectable contaminating nonspecific nucleases. This was shown by extensive incubation (4 h) of the pooled fractions with phage A DNA. The sharpness of the bands produced after gel electrophoresis was not affected by this extensive incubation. Fraction V was further purified by DEAE-
TABLE 1. Purification of EndoR * NgoII Fractiona
Vol (ml)
Total protein (mg)
Total U
Sp act (U/ Purificamg of protein)
tion
Percent re-
covery
100 345 1.0 111.8 III. (NH4)2SO4, precipitate .29 38,580 47 2.7 938 19.2 IV. Phosphocellulose chromatography 13.5 18,000 29 4.5 7.3 V. Hydroxyapatite concentration .12.2 11,380 1,556 38.8 12 0.34 5.7 VI. DEAE-cellulose chromatography 4,560 13,411 a Quantitative determinations of NgoII activity could not be made in fractions I and II. See text for
explanation.
1302
CLANTON, RIGGSBY, AND MILLER
J. BACTERIOL.
FIG. 1. Agarose gel electrophoresis patterns ofphage A DNA digested with fractions from phosphocellulose chromatography of lysates of strain MUTK1. Incubation conditions were as described in the text. After incubations, the digested DNA was subjected to gel electrophoresis in a 1.4% agarose gel and stained with ethidium bromide. NaCl concentration of the eluting buffer increases from left to right. EndoR. NgoII eluted at 0.18 M NaCl (wells 8-10). The fractions corresponding to these wells were pooled for further purification. An activity similar to that reported for EndoR.NgoI (19) eluted earlier (0.09 M NaCl, well 5).
TABLE 2. Effect of temperature on EndoR. NgoII (fraction V)
Purity of the restriction endonuclease. The purity of the restriction endonuclease was Percentage of maximal activitya determined by polyacrylamide gel electrophoresis. Amounts of 50 pl of each of fractions I-VI Temp (°C) Enzyme activwere layered onto 10% polyacrylamide gels, and, Enzyme stability' ityh after electrophoresis, the gels were stained for 4 0 NDd protein and photographed. The DEAE-cellulose 25 12 ND fraction (fraction VI) contained a single band of 35 50 100 protein (Fig. 2A). A parallel, unstained gel was 45 50 100 sliced into 2-mm segments, and each segment 55 100 6 was crushed in 0.1 ml of buffer A in separate 65
