J. Mol. Biol. (1975) 91, 121-123
LETTERSTO
THE EDITOR
A Second Specik Endonuclease from Haemophilus
aegyptius
A second restriction-like endonuclease has been partially purified from Haemophilus aegyptiw. This enzyme cleaves bacteriophage X DNA and adenovirus 2 DNA at many sites, but cleaves simian virus 40 DNA at only one site.
A number of bacterial restriction endonucleases have been isolated (Linn & Arber, 1968; Meselson & Yuan, 1968; Middleton et al., 1972; Sharp et al., 1973; Smith & Wilcox, 1970; Takanami, 1973; Takanami & Kojo, 1973; Yoshimori, 1971), and their value as a tool for studying structure and function of DNA is immediately apparent. Of particular interest are those enzymes, of which nine are now known (Middleton et al., 1972; Sharp et al., 1973; Smith &Wilcox, 1970; Takanami, 1973; Takanami & Kojo, 1973; Yoshimori, 1971), that both recognize and cleave a specific sequence of base-pairs within a DNA duplex. Their isolation is facilitated by virtue of a simple assay procedure utilizing agarose slab gel electrophoresis (B. Sugden, B. De Troy, R. J. Roberts and J. Sambrook, unpublished observations), which results in the appearance of distinctive banding patterns for these specific endonucleases. During the course of purification of the restriction enzyme endonuclease Z, from Haemophilus aegyptius (Middleton et al., 1972) (hereinafter referred to as Hae III) we detected a second endonucleolytic activity, and this report describes the partial purification and characterization of this enzyme, Hae II. H. aegyptius, ATCC 11116, was grown on brain heart infusion, supplemented with 2 pg of nicotinamide adenine dinucleotide/ml and 10 pg of hemin/ml. Cultures were harvested during late log phase and the cells (10 g) were disrupted by sonication in 1.5 volumes of a buffer containing 0.01 M-Tris.HCl (pH 7*9), 0.01 M-2-mercaptoethanol. After high-speed centrifugation (100,000 g for 60 min) the supernatant was made 1-O M in NaCl and fractionated on a column (50 cm x 2 cm) of Bio-Gel A-05m (Biorad), eluting with the same buffer containing 1-OM-Nacl. All fractions containing exonucleolytic and endonucleolytic activity were combined and brought to 70% saturation with solid (NH&SO,. The precipitate was collected and dialyzed against a buffer containing 0.01 M-potassium phosphate (pH 7*4), 0.01 >r-2-mercaptoethanol, 09001 M-EDTA, 10% glycerol (phosphocellulose buffer) and applied to a column (25 cm x O-9 cm) of phosphocellulose (Whatman, Pll) previously equilibrated with the same buffer. Elution was carried out with 200 ml of a linear gradient from 0 Y to 1.0 M-NaCl. The assay of this column is shown in Plate I. Hae II eluted between 0.40 and 0.55 M-KCl, whereas Rae III eluted between 065 and 0.9 M-KU. Endonuclease activity in the flow-through fractions was mainly due to a little Hae III which did not bind completely (however, see later). Hae II was contaminated with an exonucleolytic activity eluting between 0.35 and 0.45 M-KCl. Further purification of Hae II was achieved by chromatography on DEAEcellulose. Appropriate fractions (27 to 33) from phosphocellulose were combined and dialyzed against phosphocellulose buffer. They were applied to a column (25 cm x 0.9 cm) of DEAE-cellulose (Whatman, DE52) previously equilibrated with phosphocellulose buffer. Elution was carried out with 200 ml of a linear gradient from 0 Y to 9
121
122
R. J. ROBERTS
ET
AL.
O-3 M-KCI, and the assay of this column is shown in Plate II. Hae II eluted between 0.13 M and 0.18 ~-Kc1 and was free of contaminating exe and endonucleolytic activity. On this column Hae II and Hae III could not be adequately separated, necessitating careful choice of fractions to be combined after phosphocellulose chromatography (see Plate I). Care was required when choosing fractions containing only Hae III, as the five largest fragments produced by this enzyme are not cleaved by Hae II (see Plate III). Both endonucleases were concentrated by dialysis against phosphocellulose buffer containing 50% glycerol, and stored at -20°C. One unit of enzyme activity is defined as the amount required to completely digest 1 pg of h DNA in one hour at 37°C. No loss of activity was detected after six months. Furthermore, both enzymes may be frozen at -70°C in phosphocellulose buffer with little loss in activity (less than 10% after six months, as determined by Gtration against h DNA). The overall recovery of Hae II and Hae III could not be quantitated with respect to the amounts present after sonication, because of the difficulty of measuring these enzymes in the presence of contaminating exo and endonucleolytic activity. The yield of each, from 10 g of cells, was 10,000 units of Hae II and 50,000 units of Hae 111. In both cases digestion proceeded in a linear manner for at least 16 hours; i.e. one unit of Hae II would digest 1 pg of h DNA in one hour or 16 pg of X DNA in 16 hours. Hae II has been characterized by its action on bacteriophage h DNA, adenovirus 2 (Ad-2) DNA, and simian virus (SV40) DNA, and the results are shown in Plate III. Digests were performed with Hae II alone, Hae III alone, or a mixture of both enzymes. For X DNA and Ad-2 DNA, digestion with the mixture leads to the production of new bands, indicating that Hae II is recognizing a sequence quite distinct from that recognized by Hae III. In contrast to Hae II, which makes many breaks on SV40 DNA, Hae II cleaves SV40 DNA at only one site (Plate III). From Plate I it is apparent that SV40 DNA is converted to a linear form after treatment with Hae II, as judged by a comparison of the product with linear SV40 DNA produced by the action of Eco RI (Mulder & Delius, 1972). Furthermore, from analysis of the double digest of Hae II and Hae III on SV40 it is clear that the site of cleavage lies within the Hae III H fragment and is therefore located between positions 0.80 and O-835 on the standard SV40 map (Danna et al., 1973; P. Lebowitz, W. Siegel & J. Sklar, unpublished observations). Clearly it is possible that this enzyme is identical with one of the previously described restriction enzymes, and this possibility was tested by performing double digests with Hae II and the known restriction endonucleases. In all cases except one, Hae II was clearly different as judged by the appearance of more bands in the double digest than in either single digest, The exception was endonuclease H-l, which was clearly recognizing the same sequence as Hae II (Plate III). Unfortunately, in our hands endonuclease H-l failed to give clean banding patterns on h DNA; however, there are no bands present in the double digest which are missing in either single digest. Endonuclease H-l is chromatographically distinct from Hae II, and may well cleave at an alternate site within the recognition sequence. The exact nature of that sequence is unknown, although Takanami & Kojo (1973) have found pC and pT as the 5’.terminal nucleotides after cleavage with endonuclease H-l. In view of the previous occurrence of restriction endonucleases in the Haemophilus genus it seems likely that Hae II is indeed a restriction endonuclease; however, we have no direct evidence pertinent to this question.
PLATE I. Assay of phosphocellulose column. Samples (2.~1) of column fractions (6-ml) were incubated at .37”C for 1 h in a reaction mistw(s (.50 ~1) cont,aining 2 pg A DNA, 6 mM-Tris .HCl (pH 7.9), 6 mM-XgCl,, 6 mM-l?-mercaptoethatnol. Ijigestion was st,opped by the addition of 5 ~1 of a solut,ion containing 50,& sodium dodecyl snlfatv. 0.1 x-Na,EDTA. 15 ~1 of a solution containing 5O”/b sucrose, 0.2% bromophenol blue were added antI the samples were loaded onto a 1.4% agarose slab gel, 20 cm x 20 cm x 0.3 cm (H. Sogdrn cf rtl., unpublished experiments). Electrophorosis was carried out at 150 1’ for 1.5 h. Wands ww’ +ainctl with cthidium bromide and photographed during U.V. irradiat)ion. Fraction numbw~ an> inclicatrtl ahovr each channel. The gradient was st,artotl while fraction 9 was twing collwt~~~l.
PLATE II. Assay of DEAE-cellulose c&mm. Samples (2.~1) of column fractions were assayed as described in the legend to L’lat,o 1. Digestion was for 2 h at 37°C. Fract,ion numbers me indicat,d above each channel. The gradirnt was started while fraction 5 was being colkctrd.
I’LATE III. Agarose gel electrophoresis of entlonucleasr H-l, Har II, aud Hat III digests of A, .\d-2, and SV40 DNAs. Each DKA sample (I-pg) was digested at 37°C for 16 h in a 50.~1 reaction mixture containing and 0.1 unit of the indicated 6 mwTris.HCl (pH 7.9), 6 mM-MgCl,, 6 mM-2.mercaptoethanol, rehtriction endonuclease (1 to 5 ~1). Digestion was terminated by the addition of 0.1 M-Na,EDTA, R”,, sodium dodecyl sulfate. Each sample was adjusted to 10% sucrose, 0.025°/0 bromophsnol hluo, before loading onto a 1.4% agarose slab gel. Running conditions and staining procedures \\fsrc ewentially as previously described (Sharp et al., 1973). Slot 1, Hae III on X DN.4. 2. Hal* Hat II on X DNA. 3, Hae II on h DNA. 4, Has III on Ad-2 DNA. 5, Hat III and Hae 11 III on A4c1-2DNA. 6, Hae II on Ad-2 DNA. 7, Hae III on SV40 DNA. 8, Hae III + Hat II on RV40 I)NA. 9, Hae II on SV40 DNA. 10, Eco RI on SV40 DN.4. 11. H-l on h DN.4. 12, H-l -!- HH(L I I on h 1)NA. 13. Hae II on X DNA.
LETTERS
TO
THE
123
EDITOR
During one preparation of the endonucleases from H. aegyptiw an apparently different endonucleolytic activity was detected in the flow-through from the phosphocellulose column. So far this activity has not proved amenable to further study. However, for that reason we propose the nomenclature Hae II for the enzyme described in this report and Hae III for the previously reported endonuclease Z, in accordance with the proposal of Smith & Nathans (1973). The authors wish to thank B. Sugden, who developed the agarose slab gel techniques, and J. Sambrook for many useful discussions. This investigation was supported by a grant (CA 13106) from the National Cancer Institute. Cold Spring Harbor Cold Spring Harbor N.Y. 11724, U.S.A.
J. ROBERTS JAMES B. Bmrrrrvm~R~ NINA F. TABACHNIK~ PHYLLIS A. MYERS
Laboratory
RICHARD
t Present address: Crown College, University of California, Santa Cruz, Cal. 95060, U.S.A. $ Present address: 1776 Yale Station, New Haven, Corm. 06620, U.S.A. Received 2 July 1974, and in revised form 1Ei September
1974
REFERENCES Danna, K. J., Sack, G. & Nathans, D. (1973). J. Mol. Biol. 78, 363-376. Linn, S. & Arber, W. (1968). Proc. Nat. Ad. Sci., U.S.A. 59, 1300-1306. Meselson, M. t Yuan, R. (1968). Nature (London), 217, 1110-1114. Middleton, J. H., Edgell, M. H. & Hutchison, C. A. III (1972). J. VGoZ. 19, 42-50. Mulder, C. & Delius, H. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 3215-3219. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemdy, 12, 3055-3063. Smith, H. 0. & Nathans, D. (1973). J. Mol. BioE. 81, 419-423. Smith, H. 0. t Wilcox, K. W. (1970). J. Mol. BioZ. 57, 379-391. Takanami, M. (1973). FEBS Letters, 34, 313-322. Takanami, M. & Kojo, H. (1973). FEBS Letters, 29, 267-270. Yoshimori, R. N. (1971). Ph.D. thesis, University of California, San Francisco, U.S.A.
LETTERSTO
THE EDITOR
A Second Specik Endonuclease from Haemophilus
aegyptius
A second restriction-like endonuclease has been partially purified from Haemophilus aegyptiw. This enzyme cleaves bacteriophage X DNA and adenovirus 2 DNA at many sites, but cleaves simian virus 40 DNA at only one site.
A number of bacterial restriction endonucleases have been isolated (Linn & Arber, 1968; Meselson & Yuan, 1968; Middleton et al., 1972; Sharp et al., 1973; Smith & Wilcox, 1970; Takanami, 1973; Takanami & Kojo, 1973; Yoshimori, 1971), and their value as a tool for studying structure and function of DNA is immediately apparent. Of particular interest are those enzymes, of which nine are now known (Middleton et al., 1972; Sharp et al., 1973; Smith &Wilcox, 1970; Takanami, 1973; Takanami & Kojo, 1973; Yoshimori, 1971), that both recognize and cleave a specific sequence of base-pairs within a DNA duplex. Their isolation is facilitated by virtue of a simple assay procedure utilizing agarose slab gel electrophoresis (B. Sugden, B. De Troy, R. J. Roberts and J. Sambrook, unpublished observations), which results in the appearance of distinctive banding patterns for these specific endonucleases. During the course of purification of the restriction enzyme endonuclease Z, from Haemophilus aegyptius (Middleton et al., 1972) (hereinafter referred to as Hae III) we detected a second endonucleolytic activity, and this report describes the partial purification and characterization of this enzyme, Hae II. H. aegyptius, ATCC 11116, was grown on brain heart infusion, supplemented with 2 pg of nicotinamide adenine dinucleotide/ml and 10 pg of hemin/ml. Cultures were harvested during late log phase and the cells (10 g) were disrupted by sonication in 1.5 volumes of a buffer containing 0.01 M-Tris.HCl (pH 7*9), 0.01 M-2-mercaptoethanol. After high-speed centrifugation (100,000 g for 60 min) the supernatant was made 1-O M in NaCl and fractionated on a column (50 cm x 2 cm) of Bio-Gel A-05m (Biorad), eluting with the same buffer containing 1-OM-Nacl. All fractions containing exonucleolytic and endonucleolytic activity were combined and brought to 70% saturation with solid (NH&SO,. The precipitate was collected and dialyzed against a buffer containing 0.01 M-potassium phosphate (pH 7*4), 0.01 >r-2-mercaptoethanol, 09001 M-EDTA, 10% glycerol (phosphocellulose buffer) and applied to a column (25 cm x O-9 cm) of phosphocellulose (Whatman, Pll) previously equilibrated with the same buffer. Elution was carried out with 200 ml of a linear gradient from 0 Y to 1.0 M-NaCl. The assay of this column is shown in Plate I. Hae II eluted between 0.40 and 0.55 M-KCl, whereas Rae III eluted between 065 and 0.9 M-KU. Endonuclease activity in the flow-through fractions was mainly due to a little Hae III which did not bind completely (however, see later). Hae II was contaminated with an exonucleolytic activity eluting between 0.35 and 0.45 M-KCl. Further purification of Hae II was achieved by chromatography on DEAEcellulose. Appropriate fractions (27 to 33) from phosphocellulose were combined and dialyzed against phosphocellulose buffer. They were applied to a column (25 cm x 0.9 cm) of DEAE-cellulose (Whatman, DE52) previously equilibrated with phosphocellulose buffer. Elution was carried out with 200 ml of a linear gradient from 0 Y to 9
121
122
R. J. ROBERTS
ET
AL.
O-3 M-KCI, and the assay of this column is shown in Plate II. Hae II eluted between 0.13 M and 0.18 ~-Kc1 and was free of contaminating exe and endonucleolytic activity. On this column Hae II and Hae III could not be adequately separated, necessitating careful choice of fractions to be combined after phosphocellulose chromatography (see Plate I). Care was required when choosing fractions containing only Hae III, as the five largest fragments produced by this enzyme are not cleaved by Hae II (see Plate III). Both endonucleases were concentrated by dialysis against phosphocellulose buffer containing 50% glycerol, and stored at -20°C. One unit of enzyme activity is defined as the amount required to completely digest 1 pg of h DNA in one hour at 37°C. No loss of activity was detected after six months. Furthermore, both enzymes may be frozen at -70°C in phosphocellulose buffer with little loss in activity (less than 10% after six months, as determined by Gtration against h DNA). The overall recovery of Hae II and Hae III could not be quantitated with respect to the amounts present after sonication, because of the difficulty of measuring these enzymes in the presence of contaminating exo and endonucleolytic activity. The yield of each, from 10 g of cells, was 10,000 units of Hae II and 50,000 units of Hae 111. In both cases digestion proceeded in a linear manner for at least 16 hours; i.e. one unit of Hae II would digest 1 pg of h DNA in one hour or 16 pg of X DNA in 16 hours. Hae II has been characterized by its action on bacteriophage h DNA, adenovirus 2 (Ad-2) DNA, and simian virus (SV40) DNA, and the results are shown in Plate III. Digests were performed with Hae II alone, Hae III alone, or a mixture of both enzymes. For X DNA and Ad-2 DNA, digestion with the mixture leads to the production of new bands, indicating that Hae II is recognizing a sequence quite distinct from that recognized by Hae III. In contrast to Hae II, which makes many breaks on SV40 DNA, Hae II cleaves SV40 DNA at only one site (Plate III). From Plate I it is apparent that SV40 DNA is converted to a linear form after treatment with Hae II, as judged by a comparison of the product with linear SV40 DNA produced by the action of Eco RI (Mulder & Delius, 1972). Furthermore, from analysis of the double digest of Hae II and Hae III on SV40 it is clear that the site of cleavage lies within the Hae III H fragment and is therefore located between positions 0.80 and O-835 on the standard SV40 map (Danna et al., 1973; P. Lebowitz, W. Siegel & J. Sklar, unpublished observations). Clearly it is possible that this enzyme is identical with one of the previously described restriction enzymes, and this possibility was tested by performing double digests with Hae II and the known restriction endonucleases. In all cases except one, Hae II was clearly different as judged by the appearance of more bands in the double digest than in either single digest, The exception was endonuclease H-l, which was clearly recognizing the same sequence as Hae II (Plate III). Unfortunately, in our hands endonuclease H-l failed to give clean banding patterns on h DNA; however, there are no bands present in the double digest which are missing in either single digest. Endonuclease H-l is chromatographically distinct from Hae II, and may well cleave at an alternate site within the recognition sequence. The exact nature of that sequence is unknown, although Takanami & Kojo (1973) have found pC and pT as the 5’.terminal nucleotides after cleavage with endonuclease H-l. In view of the previous occurrence of restriction endonucleases in the Haemophilus genus it seems likely that Hae II is indeed a restriction endonuclease; however, we have no direct evidence pertinent to this question.
PLATE I. Assay of phosphocellulose column. Samples (2.~1) of column fractions (6-ml) were incubated at .37”C for 1 h in a reaction mistw(s (.50 ~1) cont,aining 2 pg A DNA, 6 mM-Tris .HCl (pH 7.9), 6 mM-XgCl,, 6 mM-l?-mercaptoethatnol. Ijigestion was st,opped by the addition of 5 ~1 of a solut,ion containing 50,& sodium dodecyl snlfatv. 0.1 x-Na,EDTA. 15 ~1 of a solution containing 5O”/b sucrose, 0.2% bromophenol blue were added antI the samples were loaded onto a 1.4% agarose slab gel, 20 cm x 20 cm x 0.3 cm (H. Sogdrn cf rtl., unpublished experiments). Electrophorosis was carried out at 150 1’ for 1.5 h. Wands ww’ +ainctl with cthidium bromide and photographed during U.V. irradiat)ion. Fraction numbw~ an> inclicatrtl ahovr each channel. The gradient was st,artotl while fraction 9 was twing collwt~~~l.
PLATE II. Assay of DEAE-cellulose c&mm. Samples (2.~1) of column fractions were assayed as described in the legend to L’lat,o 1. Digestion was for 2 h at 37°C. Fract,ion numbers me indicat,d above each channel. The gradirnt was started while fraction 5 was being colkctrd.
I’LATE III. Agarose gel electrophoresis of entlonucleasr H-l, Har II, aud Hat III digests of A, .\d-2, and SV40 DNAs. Each DKA sample (I-pg) was digested at 37°C for 16 h in a 50.~1 reaction mixture containing and 0.1 unit of the indicated 6 mwTris.HCl (pH 7.9), 6 mM-MgCl,, 6 mM-2.mercaptoethanol, rehtriction endonuclease (1 to 5 ~1). Digestion was terminated by the addition of 0.1 M-Na,EDTA, R”,, sodium dodecyl sulfate. Each sample was adjusted to 10% sucrose, 0.025°/0 bromophsnol hluo, before loading onto a 1.4% agarose slab gel. Running conditions and staining procedures \\fsrc ewentially as previously described (Sharp et al., 1973). Slot 1, Hae III on X DN.4. 2. Hal* Hat II on X DNA. 3, Hae II on h DNA. 4, Has III on Ad-2 DNA. 5, Hat III and Hae 11 III on A4c1-2DNA. 6, Hae II on Ad-2 DNA. 7, Hae III on SV40 DNA. 8, Hae III + Hat II on RV40 I)NA. 9, Hae II on SV40 DNA. 10, Eco RI on SV40 DN.4. 11. H-l on h DN.4. 12, H-l -!- HH(L I I on h 1)NA. 13. Hae II on X DNA.
LETTERS
TO
THE
123
EDITOR
During one preparation of the endonucleases from H. aegyptiw an apparently different endonucleolytic activity was detected in the flow-through from the phosphocellulose column. So far this activity has not proved amenable to further study. However, for that reason we propose the nomenclature Hae II for the enzyme described in this report and Hae III for the previously reported endonuclease Z, in accordance with the proposal of Smith & Nathans (1973). The authors wish to thank B. Sugden, who developed the agarose slab gel techniques, and J. Sambrook for many useful discussions. This investigation was supported by a grant (CA 13106) from the National Cancer Institute. Cold Spring Harbor Cold Spring Harbor N.Y. 11724, U.S.A.
J. ROBERTS JAMES B. Bmrrrrvm~R~ NINA F. TABACHNIK~ PHYLLIS A. MYERS
Laboratory
RICHARD
t Present address: Crown College, University of California, Santa Cruz, Cal. 95060, U.S.A. $ Present address: 1776 Yale Station, New Haven, Corm. 06620, U.S.A. Received 2 July 1974, and in revised form 1Ei September
1974
REFERENCES Danna, K. J., Sack, G. & Nathans, D. (1973). J. Mol. Biol. 78, 363-376. Linn, S. & Arber, W. (1968). Proc. Nat. Ad. Sci., U.S.A. 59, 1300-1306. Meselson, M. t Yuan, R. (1968). Nature (London), 217, 1110-1114. Middleton, J. H., Edgell, M. H. & Hutchison, C. A. III (1972). J. VGoZ. 19, 42-50. Mulder, C. & Delius, H. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 3215-3219. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Biochemdy, 12, 3055-3063. Smith, H. 0. & Nathans, D. (1973). J. Mol. BioE. 81, 419-423. Smith, H. 0. t Wilcox, K. W. (1970). J. Mol. BioZ. 57, 379-391. Takanami, M. (1973). FEBS Letters, 34, 313-322. Takanami, M. & Kojo, H. (1973). FEBS Letters, 29, 267-270. Yoshimori, R. N. (1971). Ph.D. thesis, University of California, San Francisco, U.S.A.
