Plant Molecular Biology 18: 865-871, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
865
Purification and properties of DNA topoisomerase I from broccoli Joseph J. Kieber 1, Mary F. Lopez 2, Alain F. Tissier and Ethan Signer*
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA (*author for correspondence)," Present addresses." 1Plant Science Institute, Department of Biology, University of Pennsylvania, PA 19104-6018, USA ; 2MzTlipore Corporation, Bedford, MA 01730, USA Received 28 August 1992; accepted in revised form 19 November 1991
Key words: broccoli, topoisomerase, protein purification
Abstract We have purified a topoisomerase activity from broccoli (Brassica oleracea var. italica) to near homogeneity. The enzyme is an 80 kDa monomer as judged by gel filtration chromatography and SDS gel electrophoresis, though it may represent a proteolytic fragment of a larger protein. The enzyme is capable of removing both negative and positive supercoils in steps of one, does not absolutely require Mg 2 +, is only very weakly stimulated by NaC1, is inhibited by camptothecin, and cross-reacts with an antibody directed against human D N A topoisomerase I. These properties identify the enzyme as a eukaryotic type I topoisomerase.
Introduction DNA topoisomerases have been implicated in a host of processes affecting D N A including transcription, replication and recombination [29, 30]. Topoisomerase I, which changes the linking number of D N A in increment of one by the transient breakage of a single strand, has been isolated from several plant sources [2, 6, 7, 12, 20, 25]. These appear to be typical eukaryotic type I enzymes, except for the chloroplast enzyme which appears to be more similar to prokaryotic topoisomerase I. Fukata et al. [ 10, 11 ] have reported partial purification of a type I topoisomerase from Brassica oleracea var. botrytis (cauliflower). Here we describe the purification to near homogeneity of a type I topoisomerase from the closely related crucifer Brassica oleracea var. italica (broccoli). The
enzyme appears to be an 80 kDa protein with properties similar to other eukaryotic type ! topoisomerases.
Materials and methods S-Sepharose, MonoS, MonoQ and Superose 6 were from Pharmacia/LKB. All columns were run with an FPLC apparatus from Pharmacia. Phosphocellulose P11 was from Whatman. The human anti-topoisomerase I antibody (AFCDC9) was from the Center for Disease Control, Atlanta, Georgia. Supercoiled p S K - (Stratagene) was purified on a CsC1 gradient as described [16]. Camptothecin, adriamycin, ellipticine and berenil were from Sigma Chemical Co. Etoposide, teniposide, and m-AMSA were kindly supplied by the Division of Cancer Treatment, the National
866 Cancer Institute. Calf thymus topoisomerase I was from Bethesda Research Labs. Radiochemicals were from New England Nuclear. The standard assay mixture contained 0.5 mg supercoiled p S K - , 50mM Tris-HC1 pH 7.5, 5mM MgC12, 1 mM DTT, 50#g/ml bovine serum albumin, and 50 mM KC1 in 15 #1. After incubation at 37 °C for 15 min, the reaction was stopped with 3 #1 10~o sodium dodecyl sulfate (SDS), 20~o glycerol and 0.5 #g/ml bromphenol blue. The sample was electrophoresed on an 0.8 ~o agarose gel, for 2 h at 100 V, stained with ethidium bromide (EtBr) and photographed. One unit of enzyme is defined as the amount capable of relaxing 0.5 #g of supercoiled DNA in 15 min. For relaxation of positively supercoiled DNA, pSK- was relaxed to completion with calf thymus topoisomerase I, extracted with an equal volume of phenol/CHC13, precipitated with ethanol, resuspended in H20 and then assayed in the presence of varying amounts of EtBr. SDS-polyacrylamide electrophoresis was done in a discontinuous system [15] and stained with silver [17]. Western blots were performed as described [27]. Enzyme purification is shown in Fig. 1. All operations were done at 4 ° C. 50 heads of broccoli were passed twice through a Champion juicer (model G5-N6-853S) with constant addition per head o f ca. 200 ml Honda buffer (0.44 M sucrose, 1.25~o Ficoll, 2.5~/o Dextran T40, 20 mM 4-(2hydroxyethyl)-1-piper azineethanesulfonic acid (HEPES) pH 7.4, 10 mM MgC12, 0.5~o Triton X-100, 0.5 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonylfluoride (PMSF)). The suspension was filtered through two layers of Miracloth (Calbiochem) and the nuclei pelleted at 8000 rpm in a Sorvall GS-3 rotor. The pellets were resuspended in 1 litre of HEG buffer (25mM HEPES pH7.4, 1 mM EDTA, 15~o glycerol, 1 mM DTT, 0.7 #g/ml Pepstatin A, 0.5 #g/ml leupeptin and 0.5 mM PMSF). NaC1 was added to a final concentration of 1 M, and the viscous solution was occasionally swirled vigorously by hand for 10 min. One-fifth volume of polyethylene glycol (PEG) solution (50~o w/v PEG 8000 in H20) was then added, and the mix-
50 Heads of Broeolli
grind with juicer [n buffer + Triton X-100
~
Honiogenate efg 9kTpmX 10'
7"
865
Purification and properties of DNA topoisomerase I from broccoli Joseph J. Kieber 1, Mary F. Lopez 2, Alain F. Tissier and Ethan Signer*
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA (*author for correspondence)," Present addresses." 1Plant Science Institute, Department of Biology, University of Pennsylvania, PA 19104-6018, USA ; 2MzTlipore Corporation, Bedford, MA 01730, USA Received 28 August 1992; accepted in revised form 19 November 1991
Key words: broccoli, topoisomerase, protein purification
Abstract We have purified a topoisomerase activity from broccoli (Brassica oleracea var. italica) to near homogeneity. The enzyme is an 80 kDa monomer as judged by gel filtration chromatography and SDS gel electrophoresis, though it may represent a proteolytic fragment of a larger protein. The enzyme is capable of removing both negative and positive supercoils in steps of one, does not absolutely require Mg 2 +, is only very weakly stimulated by NaC1, is inhibited by camptothecin, and cross-reacts with an antibody directed against human D N A topoisomerase I. These properties identify the enzyme as a eukaryotic type I topoisomerase.
Introduction DNA topoisomerases have been implicated in a host of processes affecting D N A including transcription, replication and recombination [29, 30]. Topoisomerase I, which changes the linking number of D N A in increment of one by the transient breakage of a single strand, has been isolated from several plant sources [2, 6, 7, 12, 20, 25]. These appear to be typical eukaryotic type I enzymes, except for the chloroplast enzyme which appears to be more similar to prokaryotic topoisomerase I. Fukata et al. [ 10, 11 ] have reported partial purification of a type I topoisomerase from Brassica oleracea var. botrytis (cauliflower). Here we describe the purification to near homogeneity of a type I topoisomerase from the closely related crucifer Brassica oleracea var. italica (broccoli). The
enzyme appears to be an 80 kDa protein with properties similar to other eukaryotic type ! topoisomerases.
Materials and methods S-Sepharose, MonoS, MonoQ and Superose 6 were from Pharmacia/LKB. All columns were run with an FPLC apparatus from Pharmacia. Phosphocellulose P11 was from Whatman. The human anti-topoisomerase I antibody (AFCDC9) was from the Center for Disease Control, Atlanta, Georgia. Supercoiled p S K - (Stratagene) was purified on a CsC1 gradient as described [16]. Camptothecin, adriamycin, ellipticine and berenil were from Sigma Chemical Co. Etoposide, teniposide, and m-AMSA were kindly supplied by the Division of Cancer Treatment, the National
866 Cancer Institute. Calf thymus topoisomerase I was from Bethesda Research Labs. Radiochemicals were from New England Nuclear. The standard assay mixture contained 0.5 mg supercoiled p S K - , 50mM Tris-HC1 pH 7.5, 5mM MgC12, 1 mM DTT, 50#g/ml bovine serum albumin, and 50 mM KC1 in 15 #1. After incubation at 37 °C for 15 min, the reaction was stopped with 3 #1 10~o sodium dodecyl sulfate (SDS), 20~o glycerol and 0.5 #g/ml bromphenol blue. The sample was electrophoresed on an 0.8 ~o agarose gel, for 2 h at 100 V, stained with ethidium bromide (EtBr) and photographed. One unit of enzyme is defined as the amount capable of relaxing 0.5 #g of supercoiled DNA in 15 min. For relaxation of positively supercoiled DNA, pSK- was relaxed to completion with calf thymus topoisomerase I, extracted with an equal volume of phenol/CHC13, precipitated with ethanol, resuspended in H20 and then assayed in the presence of varying amounts of EtBr. SDS-polyacrylamide electrophoresis was done in a discontinuous system [15] and stained with silver [17]. Western blots were performed as described [27]. Enzyme purification is shown in Fig. 1. All operations were done at 4 ° C. 50 heads of broccoli were passed twice through a Champion juicer (model G5-N6-853S) with constant addition per head o f ca. 200 ml Honda buffer (0.44 M sucrose, 1.25~o Ficoll, 2.5~/o Dextran T40, 20 mM 4-(2hydroxyethyl)-1-piper azineethanesulfonic acid (HEPES) pH 7.4, 10 mM MgC12, 0.5~o Triton X-100, 0.5 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonylfluoride (PMSF)). The suspension was filtered through two layers of Miracloth (Calbiochem) and the nuclei pelleted at 8000 rpm in a Sorvall GS-3 rotor. The pellets were resuspended in 1 litre of HEG buffer (25mM HEPES pH7.4, 1 mM EDTA, 15~o glycerol, 1 mM DTT, 0.7 #g/ml Pepstatin A, 0.5 #g/ml leupeptin and 0.5 mM PMSF). NaC1 was added to a final concentration of 1 M, and the viscous solution was occasionally swirled vigorously by hand for 10 min. One-fifth volume of polyethylene glycol (PEG) solution (50~o w/v PEG 8000 in H20) was then added, and the mix-
50 Heads of Broeolli
grind with juicer [n buffer + Triton X-100
~
Honiogenate efg 9kTpmX 10'
7"
