/ . Biochem., 80, 1457-1460 (1976)
PRELIMINARY COMMUNICATION
The Site-specific Deoxyribonuclease from Bacillus pumilus
Shukuko IKAWA, Takehiko SHIBATA, and Tadahiko ANDO Laboratory of Microbiology, The Institute of Physical and Chemical Research, Wako, Saitama 351 Received for publication, October 5, 1976
A new site-specific endonuclease (DNase) was isolated from the cells of Bacillus pumilus AHU 1387 strain. This enzyme (endonuclease K.Bpu 1387) introduced double-stranded scissions at unique sites on DNA's of coli phage X, /idvl, coli phage T7, Bacillus phage 01O5C, Bacillus phage SP10, and Simian Virus 40, in the presence of magnesium ion. The activity was stimulated by the presence of NaCl.
"We have previously found site-specific "DNase activity in Bacillus pumilus AHU 1387 during a systematic study on the distribution of this type of enzyme in Bacillus genus ( i ) . In this communication, we will describe the purification and some characteristics of this site-specific DNase, endonuclease TLBpu 1387 (referred to as Endo.R.Bpu in this communication). Endo.K.Bpu was prepared as follows: bacterial cells (10 g) harvested from an early stationary phase culture in CI medium (2), were suspended in 20 ml of Buffer A (3), treated with lysozyme and disrupted by sonication, then the streptomycin supernatant fraction (Fraction II) was prepared (4). The materials precipitated from Fraction II by addition of ammonium sulfate between 35% and 6 5 % saturation were dissolved in and dialyzed against a sufficient volume of Buffer A (Fraction III). This fraction (50 mg of protein) was loaded onto a column of phosphocellulose (1x12 cm). The column was washed with 40 ml of Buffer A and cluted with a 0-1 M NaCl linear gradient in Buffer A. A reaction mixture (30 fi\) containing 0.1 (xg DNA, 50 mM Tris-HCl buffer (pH 7.5), 0.2 mM EDTA, 15 mM MgCI,, 50 mM NaCl, 2 mM 2Vol. 80, No. 6, 1976
1457
mercaptoethanol and enzyme preparation was incubated at 37° for 50 min. Treated DNA samples were analyzed by agarose gel (0.7 %) electrophoresis as described previously (3, 4). Endo.R.fl/ju was eluted from the column between 0.4 and 0.5 M NaCl (Fig. 1). The activity index [i.e., the minimal amount of protein (jtg) required for complete digestion of 0.1 fig of X D N A under the standard conditions (5) ] of this fraction was about 5. This endonuclease behaved as an enzyme in phosphocellulose- and DEAE-cellulose chromatographies and on Ultrogel AcA34 (LKB) gel-filtration (data not shown). As shown in Fig. 2, this enzyme cleaves various DNA's at unique sites. DNA from coli phage X is cleaved at about six sites to produce seven fragments, two of which are probably at the same position in the gel electrophoretic profile (the second band from the top) (Fig. 2-8). DNA from Bacillus phage 01O5C grown on either B. subtilis 168 or B. amyloliquefaciens N exhibited six bands in the profile (Fig. 2-14-16) and these host strains did not affect the results. The second band from the top of the profile contains fragments from both terminal positions of the phage DNA, which are
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
(Endonuclease
1458
PRELIMINARY COMMUNICATION
connected via cohesive ends, since when the products of the endonuclease treatment were heated at 65° for 5 min, the second band disappeared and the third and fourth bands become thicker (Fig. 2-17). The fourth band appears to contain two species of fragments, judging from the thickness of the band, and the number of cleavage sites by Endo.R.Bpu on phage 01O5C DNA is probably five. The sum of
12
3 4 5
8 9 10 11 12 L 1 3 1 4 1 5 1 6 1 7 1 8 L
the roughly estimated molecular weights of the fragments (2.59 x 10') agrees well with the molecular weight of whole DNA (2.5 X107). X dvl DNA has one site cleaved by the enzyme (Fig. 2-10). When treated SV40 DNA was analyzed two main bands appeared (Fig. 2-2). Judging from the approximate molecular weights of the fragments (2.3 X10* and 0.7 x 10s) and that of the whole molecule
Fig. 2. Cleavage of DNA's with endonuclease R. BpuUil. 1, untreated SV40 DNA; 2, treated SV4O DNA; 3, untreated ColEI DNA; 4, treated ColEI DNA; 5, untreated phage T7 DNA; 6, treated phageT7 DNA; 7, untreated phage X DNA; 8, treated phage i DNA; 9, untreated /tdvl DNA; 10, treated ;dvl DNA; 11, untreated phage SP10 DNA; 12, treated phage SP10 DNA; 13, untreated phage ^105CDNA^ 14 treated DNA from phage 01O5C grown on B. subtilis 168 (01O5C-168); 15, treated DNA from phage j41O5C grown on B. amyloliquefadens N (01O5C-N); 16, untreated phage {S105C DNA; 17, phage ?S105C-168 DNA treated with Endo.RJBpu; 18, phage 01050168 DNA treated with Endo.R-.5fpH and then heated at 65° for 5 min. The heated sample was cooled quickly: L, Z DNA treated with Endo.R.£coRI.
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
Fig. 1. Phosphocellulose chromatography of Fraction m (ammonium sulfate fraction), i DNAXwas treated with 10 ft\ of samples and analyzed by gel electrophoresis. O, protein concentration; , NaCl concentration. Photograph of gel electrophoretic profiles of the treated k DNA. Samples migrated from top to bottom.
I THE SITE-SPECIFIC DNase FROM B. pumilus 1
2 3 4 5 6 7 8 9
(3.6 x 10"), SV40 DNA is cleaved at three sites by the enzyme. The treated coli phage T7 DNA produced one band in the electrophoretic profile (Fig. 2-6). The cleavage site on T7 DNA seems to be located near the middle of the molecule. Bacillus phage SP10 DNA was cleaved at several sites (Fig. 2-12). Neither Bacillus phage SPP1 nor colicinogenic factor ColEI DNA was attacked by the enzyme (data not shown, and Fig. 2-4). Endo.R.Bpu requires only magnesium ions for the activity and the optimal concentration of MgCl, for the activity is 10 to 20 mM (Fig. 3). Magnesium ions can be partially replaced by 10 mM manganese ions but not by calcium ions (Fig. 3). The specificity of the enzyme is probably not affected by these metallic ions (Fig. 3). As shown in Fig. 4, Endo. R.Bpu requires NaCl (0.1 to 0.2 M) for activity, especially at low MgCl, concentration (4 ITIM), but the activity is inhibited by high NaCl concentrations. This enzyme exhibited nearly unchanged activity between pH 7.2 and pH 8.8 (data not shown). Endo.R.Bpu is a classical restriction enzyme, since X DNA which was previously modified in vitro in the presence of S-adenosyl-L-methionine with Fraction II from B.pumilus AHU 1387 became resistant to Endo.R.Bpu (data not shown). Vol. 80, No. 6, 1976
1 2
3
4
5
6
Fig.4. Effect of NaCl on the activity of endonuclease R.£pwl387. I DNA was treated with Endo.R.Bpu (25 fig of protein) under the standard conditions but with 4 mM MgCl,. NaCl concentrations were as follows. 1, without NaCl; 2, 10 mM; 3, 50ITIM; 4, 0.1 M; 5, 0.2 M; 6, 0.4 M. As for the sequence specificity, Endo.K.Bpu differs from endonucleases R.Alu I, R.BamHI,
R.BamNl, R.BamNx, R.BsuR, R.EcoRI, R.EcoRH, R.HaeU, R.HaeHI, R.HapU, R.HhaU, R.Hpal, R.HpaH, and R.Hphl, because the numbers of cleavages introduced in ^DNA, >idvl DNA, and SV40 DNA by these endonucleases are different from those by Endo.R.Bpu (3-16). Studies are in progress on the nucleotide sequences around the sites cleaved by Endo.R.Bpu. Note added in proof: When X DNA cleaved by Endo.R.Bpu was heated at 65° for 5 min a new band was detected below the lowest band shown in Figs. 2-8. The approximate molecular weight of the fragment is 2 x 156. We thank Drs. Y. Sasaki, K. Matsubara, and N. Yamaguchi for generous gifts of materials. This work was supported by a grant for studies on "Life science" at this Institute and in part by a grant from Miles Laboratories, Inc. REFERENCES 1. Shibata, T., Ikawa, S., Kim, C , & Ando, T. (1976) / . Bacteriol. 128, 473^*76 2. Shibata, T. & Saito, H. (1973) Mutation Res. 20, 159-173 3. Shibata, T. & Ando, T. (1976) Biochim. Biophys.
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
Fig. 3. Effects of divalent cations on the activity of [ endonuclease R.flpul387. X DNA was treated with Endo.R.Bpw (2.5 p% of protein) under the standard conditions but with the indicated divalent cations. 1, 10 mM CaCl,; 2, lOmM MnCl,; 3, without divalent cations; 4, 5mM MgCU; 5, 10mM MgCl,; 6, 20mM MgCl,; 7, 40 mM MgCl,; 8, 80 mM MgCl,; 9, untreated ; DNA.
1459
1460
11. 12. 13. 14. 15. 16.
J., & Goodman, H.M. (1973) Nature New Biol. 244, 40-43 Roberts, R.J., Breitmeyer, J.B., Tabachnik, N.F., & Myers, P.A. (1975) / . Mol. Biol. 91, 121-123 Yang, R.C., van de Voorde, A., & Fiers, W. (1976) Eur. J. Biochem. 61, 101-117 Sugisaki, H. & Takanami, M. (1973) Nature New Biol. 246, 138-140 Roberts, R.J., Myers, P.A., Morrison, A., & Murray, K. (1976) / . Mol. Biol. 103, 199-208 Sharp, P.A., Sugden, B., & Sambrook, J. (1973) Biochemistry 12, 3055-3063 Kleid, D., Humayun, Z., Jeffrey, A., & Ptashne, M. (1976) Proc. Natl. Acad. Sci. U.S. 73, 293-297
/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
Ada 442, 184-196 4. Shibata, T. & Ando, T. (1975) Mol. Gen. Genet. 138, 269-279 5. Roberts, R.J., Myers, P.A., Morrison, A., & Murray, K. (1976) / . Mol. Biol. 102, 157-165 6. Yang, R.C., van de Voorde, A., & Fiers, W. (1976) Eur. J. Biochem. 61, 119-138 7. Wilson, G.A. & Young, F.E. (1975) / . Mol. Biol. 97, 123-125 8. Bron, S., Murray, K., & Trautner, T.A. (1975) Mol. Gen. Genet. 143, 13-23 9. Streeck, R.E. & Hobom, G. (1975) Eur. J. Biochem. 57, 595-606 10. Boyer, H.W., Chow, L.T., Dugaiczyk, A., Hedgpeth,
PRELIMINARY COMMUNICATION
PRELIMINARY COMMUNICATION
The Site-specific Deoxyribonuclease from Bacillus pumilus
Shukuko IKAWA, Takehiko SHIBATA, and Tadahiko ANDO Laboratory of Microbiology, The Institute of Physical and Chemical Research, Wako, Saitama 351 Received for publication, October 5, 1976
A new site-specific endonuclease (DNase) was isolated from the cells of Bacillus pumilus AHU 1387 strain. This enzyme (endonuclease K.Bpu 1387) introduced double-stranded scissions at unique sites on DNA's of coli phage X, /idvl, coli phage T7, Bacillus phage 01O5C, Bacillus phage SP10, and Simian Virus 40, in the presence of magnesium ion. The activity was stimulated by the presence of NaCl.
"We have previously found site-specific "DNase activity in Bacillus pumilus AHU 1387 during a systematic study on the distribution of this type of enzyme in Bacillus genus ( i ) . In this communication, we will describe the purification and some characteristics of this site-specific DNase, endonuclease TLBpu 1387 (referred to as Endo.R.Bpu in this communication). Endo.K.Bpu was prepared as follows: bacterial cells (10 g) harvested from an early stationary phase culture in CI medium (2), were suspended in 20 ml of Buffer A (3), treated with lysozyme and disrupted by sonication, then the streptomycin supernatant fraction (Fraction II) was prepared (4). The materials precipitated from Fraction II by addition of ammonium sulfate between 35% and 6 5 % saturation were dissolved in and dialyzed against a sufficient volume of Buffer A (Fraction III). This fraction (50 mg of protein) was loaded onto a column of phosphocellulose (1x12 cm). The column was washed with 40 ml of Buffer A and cluted with a 0-1 M NaCl linear gradient in Buffer A. A reaction mixture (30 fi\) containing 0.1 (xg DNA, 50 mM Tris-HCl buffer (pH 7.5), 0.2 mM EDTA, 15 mM MgCI,, 50 mM NaCl, 2 mM 2Vol. 80, No. 6, 1976
1457
mercaptoethanol and enzyme preparation was incubated at 37° for 50 min. Treated DNA samples were analyzed by agarose gel (0.7 %) electrophoresis as described previously (3, 4). Endo.R.fl/ju was eluted from the column between 0.4 and 0.5 M NaCl (Fig. 1). The activity index [i.e., the minimal amount of protein (jtg) required for complete digestion of 0.1 fig of X D N A under the standard conditions (5) ] of this fraction was about 5. This endonuclease behaved as an enzyme in phosphocellulose- and DEAE-cellulose chromatographies and on Ultrogel AcA34 (LKB) gel-filtration (data not shown). As shown in Fig. 2, this enzyme cleaves various DNA's at unique sites. DNA from coli phage X is cleaved at about six sites to produce seven fragments, two of which are probably at the same position in the gel electrophoretic profile (the second band from the top) (Fig. 2-8). DNA from Bacillus phage 01O5C grown on either B. subtilis 168 or B. amyloliquefaciens N exhibited six bands in the profile (Fig. 2-14-16) and these host strains did not affect the results. The second band from the top of the profile contains fragments from both terminal positions of the phage DNA, which are
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
(Endonuclease
1458
PRELIMINARY COMMUNICATION
connected via cohesive ends, since when the products of the endonuclease treatment were heated at 65° for 5 min, the second band disappeared and the third and fourth bands become thicker (Fig. 2-17). The fourth band appears to contain two species of fragments, judging from the thickness of the band, and the number of cleavage sites by Endo.R.Bpu on phage 01O5C DNA is probably five. The sum of
12
3 4 5
8 9 10 11 12 L 1 3 1 4 1 5 1 6 1 7 1 8 L
the roughly estimated molecular weights of the fragments (2.59 x 10') agrees well with the molecular weight of whole DNA (2.5 X107). X dvl DNA has one site cleaved by the enzyme (Fig. 2-10). When treated SV40 DNA was analyzed two main bands appeared (Fig. 2-2). Judging from the approximate molecular weights of the fragments (2.3 X10* and 0.7 x 10s) and that of the whole molecule
Fig. 2. Cleavage of DNA's with endonuclease R. BpuUil. 1, untreated SV40 DNA; 2, treated SV4O DNA; 3, untreated ColEI DNA; 4, treated ColEI DNA; 5, untreated phage T7 DNA; 6, treated phageT7 DNA; 7, untreated phage X DNA; 8, treated phage i DNA; 9, untreated /tdvl DNA; 10, treated ;dvl DNA; 11, untreated phage SP10 DNA; 12, treated phage SP10 DNA; 13, untreated phage ^105CDNA^ 14 treated DNA from phage 01O5C grown on B. subtilis 168 (01O5C-168); 15, treated DNA from phage j41O5C grown on B. amyloliquefadens N (01O5C-N); 16, untreated phage {S105C DNA; 17, phage ?S105C-168 DNA treated with Endo.RJBpu; 18, phage 01050168 DNA treated with Endo.R-.5fpH and then heated at 65° for 5 min. The heated sample was cooled quickly: L, Z DNA treated with Endo.R.£coRI.
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
Fig. 1. Phosphocellulose chromatography of Fraction m (ammonium sulfate fraction), i DNAXwas treated with 10 ft\ of samples and analyzed by gel electrophoresis. O, protein concentration; , NaCl concentration. Photograph of gel electrophoretic profiles of the treated k DNA. Samples migrated from top to bottom.
I THE SITE-SPECIFIC DNase FROM B. pumilus 1
2 3 4 5 6 7 8 9
(3.6 x 10"), SV40 DNA is cleaved at three sites by the enzyme. The treated coli phage T7 DNA produced one band in the electrophoretic profile (Fig. 2-6). The cleavage site on T7 DNA seems to be located near the middle of the molecule. Bacillus phage SP10 DNA was cleaved at several sites (Fig. 2-12). Neither Bacillus phage SPP1 nor colicinogenic factor ColEI DNA was attacked by the enzyme (data not shown, and Fig. 2-4). Endo.R.Bpu requires only magnesium ions for the activity and the optimal concentration of MgCl, for the activity is 10 to 20 mM (Fig. 3). Magnesium ions can be partially replaced by 10 mM manganese ions but not by calcium ions (Fig. 3). The specificity of the enzyme is probably not affected by these metallic ions (Fig. 3). As shown in Fig. 4, Endo. R.Bpu requires NaCl (0.1 to 0.2 M) for activity, especially at low MgCl, concentration (4 ITIM), but the activity is inhibited by high NaCl concentrations. This enzyme exhibited nearly unchanged activity between pH 7.2 and pH 8.8 (data not shown). Endo.R.Bpu is a classical restriction enzyme, since X DNA which was previously modified in vitro in the presence of S-adenosyl-L-methionine with Fraction II from B.pumilus AHU 1387 became resistant to Endo.R.Bpu (data not shown). Vol. 80, No. 6, 1976
1 2
3
4
5
6
Fig.4. Effect of NaCl on the activity of endonuclease R.£pwl387. I DNA was treated with Endo.R.Bpu (25 fig of protein) under the standard conditions but with 4 mM MgCl,. NaCl concentrations were as follows. 1, without NaCl; 2, 10 mM; 3, 50ITIM; 4, 0.1 M; 5, 0.2 M; 6, 0.4 M. As for the sequence specificity, Endo.K.Bpu differs from endonucleases R.Alu I, R.BamHI,
R.BamNl, R.BamNx, R.BsuR, R.EcoRI, R.EcoRH, R.HaeU, R.HaeHI, R.HapU, R.HhaU, R.Hpal, R.HpaH, and R.Hphl, because the numbers of cleavages introduced in ^DNA, >idvl DNA, and SV40 DNA by these endonucleases are different from those by Endo.R.Bpu (3-16). Studies are in progress on the nucleotide sequences around the sites cleaved by Endo.R.Bpu. Note added in proof: When X DNA cleaved by Endo.R.Bpu was heated at 65° for 5 min a new band was detected below the lowest band shown in Figs. 2-8. The approximate molecular weight of the fragment is 2 x 156. We thank Drs. Y. Sasaki, K. Matsubara, and N. Yamaguchi for generous gifts of materials. This work was supported by a grant for studies on "Life science" at this Institute and in part by a grant from Miles Laboratories, Inc. REFERENCES 1. Shibata, T., Ikawa, S., Kim, C , & Ando, T. (1976) / . Bacteriol. 128, 473^*76 2. Shibata, T. & Saito, H. (1973) Mutation Res. 20, 159-173 3. Shibata, T. & Ando, T. (1976) Biochim. Biophys.
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
Fig. 3. Effects of divalent cations on the activity of [ endonuclease R.flpul387. X DNA was treated with Endo.R.Bpw (2.5 p% of protein) under the standard conditions but with the indicated divalent cations. 1, 10 mM CaCl,; 2, lOmM MnCl,; 3, without divalent cations; 4, 5mM MgCU; 5, 10mM MgCl,; 6, 20mM MgCl,; 7, 40 mM MgCl,; 8, 80 mM MgCl,; 9, untreated ; DNA.
1459
1460
11. 12. 13. 14. 15. 16.
J., & Goodman, H.M. (1973) Nature New Biol. 244, 40-43 Roberts, R.J., Breitmeyer, J.B., Tabachnik, N.F., & Myers, P.A. (1975) / . Mol. Biol. 91, 121-123 Yang, R.C., van de Voorde, A., & Fiers, W. (1976) Eur. J. Biochem. 61, 101-117 Sugisaki, H. & Takanami, M. (1973) Nature New Biol. 246, 138-140 Roberts, R.J., Myers, P.A., Morrison, A., & Murray, K. (1976) / . Mol. Biol. 103, 199-208 Sharp, P.A., Sugden, B., & Sambrook, J. (1973) Biochemistry 12, 3055-3063 Kleid, D., Humayun, Z., Jeffrey, A., & Ptashne, M. (1976) Proc. Natl. Acad. Sci. U.S. 73, 293-297
/. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/80/6/1457/855726 by New York University user on 03 February 2019
Ada 442, 184-196 4. Shibata, T. & Ando, T. (1975) Mol. Gen. Genet. 138, 269-279 5. Roberts, R.J., Myers, P.A., Morrison, A., & Murray, K. (1976) / . Mol. Biol. 102, 157-165 6. Yang, R.C., van de Voorde, A., & Fiers, W. (1976) Eur. J. Biochem. 61, 119-138 7. Wilson, G.A. & Young, F.E. (1975) / . Mol. Biol. 97, 123-125 8. Bron, S., Murray, K., & Trautner, T.A. (1975) Mol. Gen. Genet. 143, 13-23 9. Streeck, R.E. & Hobom, G. (1975) Eur. J. Biochem. 57, 595-606 10. Boyer, H.W., Chow, L.T., Dugaiczyk, A., Hedgpeth,
PRELIMINARY COMMUNICATION
