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Biochemical and Biophysical Analyses of Recombinant Forms of Human Topoisomerase I (∗)

  • Lance Stewart
    Footnotes
    Affiliations
    Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242
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  • Gregory C. Ireton
    Affiliations
    Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242
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  • Leon H. Parker
    Footnotes
    Affiliations
    Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242
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  • Knut R. Madden
    Affiliations
    Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242
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  • James J. Champoux
    Correspondence
    To whom correspondence should be addressed: Dept. of Microbiology, Box 357242, School of Medicine, University of Washington, Seattle, WA 98195-7242
    Affiliations
    Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242
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  • Author Footnotes
    ∗ This work was supported by National Institutes of Health Grant GM49156. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by American Cancer Society Grant PF-3905.
    Supported by Public Health Service National Research Service Award T32-GM07207 from the NIGMS, National Institutes of Health.
      Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH2-terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine. To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH2-terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH2- and COOH-terminally truncated core fragment encompassing residues 175-659. The specific activity of the full-length and Topo70 enzymes are indistinguishable from the native human Topo I purified from HeLa cells. Each protein is inhibited by camptothecin, topotecan, and 9-amino-camptothecin, but not by ATP. Activity is stimulated by Mg2+, Ba2+, Ca2+, Mn2+, spermine, and spermidine. The magnitude of the stimulatory effect of Mg2+ is inversely proportional to the salt concentration. Furthermore, at KCl concentrations of 300 mM or greater, the addition of Mg2+ is inhibitory. The effects of Mg2+ and the polycations spermine and spermidine are partially additive, an indication that the stimulatory mechanisms of the two substances are different. Activity was strongly inhibited or abolished by Ni2+, Zn2+, Cu2+, Cd2+, and Co2+. An examination of the hydrodynamic properties of full-length Topo I, Topo70, and Topo58 demonstrates that the core, linker, and COOH-terminal domains fold into a globular structure, while the NH2-terminal domain is highly extended. A comparison of the circular dichroism spectra of full-length Topo I and Topo70 demonstrates that residues 1-174 (~21 kDa) of Topo I are largely if not completely unfolded. This observation is consistent with the fact that the NH2-terminal domain is dispensable for activity.

      INTRODUCTION

      Eukaryotic topoisomerase I (Topo I) (
      The abbreviations used are: Topo I
      topoisomerase I
      Topo70
      NH2-terminal truncation of Topo I, missing the first 174 amino acids
      Topo58
      COOH-terminal truncation of Topo70, missing the last 106 amino acids
      PPB
      potassium phosphate buffer
      HeLa S3 cells
      suspension culture-adapted human cervical carcinoma cell line (ATCC CCL 2.2)
      PEG
      polyethylene glycol
      PS
      phenyl-Sepharose
      MES
      2-(N-morpholino)ethanesulfonic acid
      Sf9 cells
      Spodoptera frugiperda cells (ATCC CRL-1711)
      BSA
      bovine serum albumin
      CAPS
      3-(cyclohexylamino)-1-propanesulfonic acid
      DTT
      dithiothreitol
      PAGE
      polyacrylamide gel electrophoresis.
      ) is capable of relaxing both negatively and positively supercoiled DNA. The enzyme catalyzes changes in the superhelical state of duplex DNA by transiently breaking a single strand, allowing for unwinding of positively supercoiled DNA or rewinding of negatively supercoiled DNA (reviewed in (
      • Champoux J.J.
      )). No metal cation or energy cofactor is required for Topo I activity, although Mg2+ and Ca2+, as well as the polycation spermidine, have been shown to stimulate activity(
      • Goto T.
      • Laipis P.
      • Wang J.C.
      ,
      • Liu L.F.
      • Miller K.G.
      ,
      • McConaughy B.L.
      • Young L.S.
      • Champoux J.J.
      ,
      • Srivenugopal K.S.
      • Morris D.R.
      ). Phosphodiester bond energy is preserved during the nicking-closing cycle by the formation of a phosphotyrosine bond between the active-site tyrosine and the 3′-end of the broken strand (
      • Champoux J.J.
      ,
      • Champoux J.J.
      ,
      • Lynn R.M.
      • Bjornsti M.-A.
      • Caron P.R.
      • Wang J.C.
      ). This covalent intermediate can be trapped by denaturing the enzyme during catalysis with either SDS or alkali(
      • Been M.D.
      • Burgess R.R.
      • Champoux J.J.
      ,
      • Champoux J.J.
      ,
      • Champoux J.J.
      ). Sequence analyses of a large number of SDS-induced breakage sites indicated that the cellular Topo I enzymes will cleave at specific sequences(
      • Been M.D.
      • Burgess R.R.
      • Champoux J.J.
      ,
      • Edwards K.A.
      • Halligan B.D.
      • Davis J.L.
      • Nivera N.L.
      • Liu L.F.
      ,
      • Tanizawa A.
      • Khon K.W.
      • Pommier Y.
      ), but there is only limited sequence similarity between such sites (
      • Been M.D.
      • Burgess R.R.
      • Champoux J.J.
      ,
      • Edwards K.A.
      • Halligan B.D.
      • Davis J.L.
      • Nivera N.L.
      • Liu L.F.
      ,
      • Tanizawa A.
      • Khon K.W.
      • Pommier Y.
      ,
      • Bonven B.J.
      • Gocke E.
      • Westergaard O.
      ,
      • Parker L.H.
      • Champoux J.J.
      ,
      • Shen C.C.
      • Shen C.-K. J.
      ,
      • Porter S.E.
      • Champoux J.J.
      ). The SDS-induced cleavage at many breakage sites is enhanced by camptothecin, a plant alkaloid that inhibits the cellular enzymes by reversibly binding to the covalent Topo I-DNA intermediate in a manner that slows the the religation step of catalysis (
      • Porter S.E.
      • Champoux J.J.
      ,
      • Champoux J.J.
      • Aronoff R.
      ,
      • Hertzberg R.P.
      • Busby R.W.
      • Caranfa M.J.
      • Holden K.G.
      • Johnson R.K.
      • Hecht S.M.
      • Kingsbury W.D.
      ,
      • Hsiang Y.-H.
      • Hertzberg R.
      • Hecht S.
      • Liu L.F.
      ,
      • Kjeldsen E.
      • Mollerup S.
      • Thomsen B.
      • Bonven B.J.
      • Bolund L.
      • Westergaard O.
      ,
      • Porter S.E.
      • Champoux J.J.
      ).
      The human Topo I is composed of 765 residues with a predicted molecular mass of 91 kDa. Sequence comparisons of cellular eukaryotic Topo I proteins demonstrate that the human Topo I can be divided into four domains (Fig. 1). (
      For further information, contact J. C. Wang at [email protected] .
      ) Residues Met1-Lys197 (24 kDa) comprise the unconserved NH2-terminal domain, which is highly charged (Asp + Glu = 27%; His + Lys + Arg = 68%) and contains four putative nuclear localization signals(
      • Alsner J.
      • Svejstrup J.Q.
      • Kjeldsen E.
      • S⊘rensen B.S.
      • Westergaard O.
      ). Residues Glu198-Ile651 (54 kDa) form the conserved core domain, which is followed by a short positively charged linker domain of unconserved residues Asp652-Glu696 (5 kDa). Finally, residues Gln697-Phe765 (8 kDa) make up the highly conserved COOH-terminal domain, which contains the active site tyrosine at position 723(
      • D'Arpa P.
      • Machlin P.S.
      • Ratrie H.I.
      • Rothfield N.F.
      • Cleveland D.W.
      • Earnshaw W.C.
      ,
      • Madden K.R.
      • Champoux J.J.
      ).2 Previous reports have demonstrated that the NH2-terminal domain is sensitive to proteolyis (
      • Liu L.F.
      • Miller K.G.
      ) and that residues 1-230 can be removed with little if any consequence for Topo I activity(
      • D'Arpa P.
      • Machlin P.S.
      • Ratrie H.I.
      • Rothfield N.F.
      • Cleveland D.W.
      • Earnshaw W.C.
      ,
      • Kikuchi A.
      • Miyaike M.
      ). In contrast, a 5-amino acid deletion from the COOH terminus abolishes activity. (
      L. Stewart, G. Ireton, and J. J. Champoux, manuscript in preparation.
      )
      Figure thumbnail gr1
      Figure 1:Domain structure of Topo I based on sequence comparisons. Based on amino acid sequence comparisons of cellular eukaryotic Topo I proteins,2 the human enzyme can be divided into four domains. Listed below each domain is the calculated molecular mass for that domain. Filled areas represent regions that are highly conserved, while open areas represent the unconserved regions. Residues Met1-Lys197 (24 kDa) comprise the unconserved amino-terminal domain, which has an unusually high percentage of negatively and positively charged residues indicated by the plus and minus signs. Black circles indicate the locations of four potential nuclear localization signals (residues Lys59-Glu65, Lys150-Asp156, Lys174-Asp180, and Lys192-Glu198)(
      • Alsner J.
      • Svejstrup J.Q.
      • Kjeldsen E.
      • S⊘rensen B.S.
      • Westergaard O.
      ). Residues Glu198-Ile651 (54-kDa) form the conserved core domain (stippled area). Residues Asp652-Glu696form an unconserved linker domain (5 kDa), which is followed by the conserved COOH-terminal domain, residues Gln697-Phe765 (8 kDa), which contains the active site tyrosine at position 723.
      We have used the baculovirus-infected insect cell system to overproduce full-length, as well as NH2- and COOH-terminal deletions of human topoisomerase I. The purified recombinant Topo I is by all biochemical criteria tested identical to the native enzyme purified from human cells. Furthermore, we find the activities of the full-length protein and an amino-terminally deleted enzyme (Topo70, missing residues 1-174) are identical in every respect. The two proteins are inhibited by camptothecin and its derivatives, are stimulated by Mg2+ with a magnitude that is inversely proportional to the salt concentration (up to 250 mM KCl), and are not affected by ATP. By comparing the circular dichroism spectra and hydrodynamic properties of Topo70 and the full-length protein, we demonstrate that the amino-terminal domain is largely if not completely unfolded, while the remainder of the molecule is relatively globular. In the accompanying paper(
      • Stewart L.
      • Ireton G.
      • Champoux J.J.
      ), we use limited proteolysis to further examine the domain structure of human Topo I.

      EXPERIMENTAL PROCEDURES

       Plasmids

      All plasmids were constructed by common subcloning techniques and propagated in either the SureTM (Stratagene) or TOP10F′ (Invitrogen) strains of Escherichia coli. Nucleotides of the cDNA clone encoding human topoisomerase I are numbered according to D'Arpa et al.(
      • D'Arpa P.
      • Machlin P.S.
      • Ratrie H.I.
      • Rothfield N.F.
      • Cleveland D.W.
      • Earnshaw W.C.
      ).

       pAc-Topo I and pAc-Topo I(Y/F)

      The wild type and active site mutant Y/F human Topo I cDNA sequences from the plasmids pKM16 and pKM18, respectively(
      • Madden K.R.
      • Champoux J.J.
      ), were inserted into the blunt-ended NheI cloning site of pBlueBac (Invitrogen) to generate pAc-Topo I and pAc-Topo I(Y/F). Restriction analyses were carried out to confirm proper orientation of the Topo I reading frames with respect to the polyhedrin gene promoter.

       pTopo70-start

      The polymerase chain reaction was used to amplify a segment of the human Topo I cDNA from position 731 to 1112. The 5′ end of the amplification product contained an XbaI restriction site followed by an initiating methionine codon immediately adjacent to the codon for residue Lys1752. The polymerase chain reaction product was digested with XbaI (which cuts 18 base pairs upstream from the initiating ATG) and NdeI (which cuts at position 901 in the Topo I cDNA) to generate a 194-base pair fragment that was subcloned into the plasmid pET11a (Novagen) to generate pTopo70-start. The subcloned sequences that had been subjected to polymerase chain reaction were sequenced to confirm proper construction of the initiation signal and to ensure that no other mutations were present.

       pET11a-Topo70

      The plasmid pTopo70-start was linearized with HindIII, which cuts the pET11a vector backbone at a site just downstream of the Topo I sequence. The HindIII 5′ overhangs were filled in with T4 DNA polymerase and all four dNTPs. This DNA was digested with NdeI, which cuts at position 901 in the Topo I coding sequences. The resulting fragment was ligated with a 2.7-kilobase pair NdeI901-NotI (blunt-ended) fragment, which contains the NdeI901-EcoRI3641 Topo I sequences followed by multiple cloning site sequences from EcoRI to NotI of the plasmid pSK+ (Stratagene). The resulting plasmid, called pET11a-Topo70, carries the coding sequence for a ~70-kDa NH2-terminally truncated form of Topo I called Topo70 that starts at a methionine immediately 5′ of Lys1752 and ends at the natural Topo I COOH terminus.

       pAc-Topo70

      The baculovirus transfer vector pBlueBac (Invitrogen) has a unique NheI cloning site, located immediately downstream of the polyhedrin gene promoter, into which we inserted an XbaI-XbaI fragment from pET11a-Topo70, which carries the entire Topo70 coding sequence. Diagnostic restriction digests were performed to confirm proper orientation of the Topo70 reading frame with respect to the polyhedrin gene promoter. The resulting plasmid is named pAc-Topo70.

       pAc-Topo70(Y/F)

      The NdeI901-AvrII2386 sequences from pKM18 (
      • Madden K.R.
      • Champoux J.J.
      ) were exchanged for the NdeI901-AvrII2386 sequences of pAc-Topo70, to generate pAc-Topo70(Y/F). This plasmid is identical to pAc-Topo70 except that it carries a mutation that converts the active site tyrosine 723 codon to a phenylalanine codon.

       pAc-Topo58

      A self-complementary NheI-stop oligonucleotide (5′-CTAGCCTAGGCCTAGG-3′) was inserted at the unique NheI2183 restriction site of the plasmid pAc-Topo70 to generate pAc-Topo58. The NheI-stop oligonucleotide introduces a diagnostic AvrII restriction site in place of the NheI site and positions a stop codon immediately 3′ of the codon for residue Ala659 of the Topo I cDNA.

       Plasmid Relaxation Assays

      Unless stated otherwise, stocks of protein were serially diluted 2-fold in standard buffer (150 mM KCl, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA, 0.1 mg/ml BSA), and reactions were initiated by the addition of 5 μl of the diluted enzyme to 15 μl of the appropriate buffer containing 0.5 μg of supercoiled pKSII+ plasmid substrate (Stratagene). The final reaction conditions are indicated in the table and figure legends. The reactions were incubated at 37°C for 10 min and terminated with 5 μl of a stop mix containing 2.5% SDS, 15% Ficoll, 0.03% bromphenol blue, 0.03% xylene cyanol, and 25 mM EDTA. The products were fractionated by 0.8% agarose gel electrophoresis and visualzed by ethidium bromide staining. The inhibitors camptothecin (Sigma), topotecan (NCI, National Institutes of Health), and 9-amino-camptothecin (NCI) were dissolved in Me2SO and stored at −20°C.

       Isolation of Recombinant Baculoviruses and Large Scale Sf9 Infection

      Recombinant baculoviruses were generated by co-transfecting Spodoptera frugiperda (Sf9) cells with linearized wild type Autographica californica multiple nucleocapsid nuclear polyhedrosis virus DNA (Invitrogen) together with transfer vector DNAs, and plaque purified according to standard procedures provided by Invitrogen. To confirm that the recombinant viruses were expressing the appropriate protein, infected cells were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Sf9 cells were maintained in TC100 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, yeastolate (3.3 g/liter), lactalbumin hydrolysate (3.3 g/liter), 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 100 units/ml of nystatin. Cells were cultured in 100 ml and 1-liter spinner flasks (Bellco) and stirred at a rate of 60 rpm in an atmosphere of 50% O2, 50% air at 27°C. The 1-liter flasks were assembled with microcarrier impellers (Bellco) that were adjusted to break the air-liquid interface. (
      S. G. Graber, personal communication.
      ) The maximum volume of medium used in the spinner flasks was 80 and 500 ml for the 100-ml and 1-liter flasks, respectively. Typically, cells were seeded at 0.5-0.8 × 106 cells/ml and cultured until the density reached 3-3.5 × 106 cells per ml (~3 days). To infect the cells, they were first pelleted by centifugation at 600 × g at room temperature for 5 min and then resuspended in fresh medium at a density of 1 × 107 cells/ml. The appropriate volume of virus stock was added to ensure a multiplicity of infection of approximately 10 plaque-forming units/cell. After stirring for 1 h at room temperature, fresh medium was added to the cells such that the final density was 3 × 106 cells/ml.

       Protein Purification

       Purification of Topo I from Baculovirus-infected Insect Cells

      All purification steps except those involving room temperature high pressure liquid chromatography (Mono Q, Mono S, and POROS columns) were carried out at 4°C. At 48 h postinfection, approximately 3 × 109 Sf9 cells were harvested by centrifugation for 5 min at 400 × g. The cells were resuspended in 1 liter of ice-cold phosphate-buffered saline and centrifuged for 5 min at 400 × g. This wash procedure was repeated twice with 250 ml of phosphate-buffered saline. The washed cells were resuspended by vigorous shaking in 40 ml of lysis buffer (50 mM KCl, 10 mM Tris-hydrochloride, pH 7.5, 2 mM MgCl2, 1% Triton X-100, 15 mM DTT, 0.15 mg/ml phenylmethylsulfonyl fluoride, 0.05 mg/ml aprotinin). The nuclei were washed twice in 80 ml of lysis buffer minus Triton X-100 and resuspended in 40 ml of resuspension buffer (50 mM KCl, 10 mM Tris-hydrochloride, pH 7.5, 2 mM MgCl2, 25 mM DTT, 0.4 mg/ml phenylmethylsulfonyl fluoride, 0.12 mg/ml aprotinin). The nuclei were adjusted to 10 mM EDTA and then lysed by the addition of 50 ml of 2 × nuclear extraction buffer (2 M NaCl, 80 mM Tris-hydrochloride, pH 7.5, 20% glycerol, 2 mM EDTA). The nuclear extract was stirred for 5 min at ~200 rpm. With continued stirring, 50 ml of polyethylene glycol (PEG) buffer (18% PEG 8000, 1 M NaCl, 10% glycerol) was added dropwise in order to precipitate the DNA(
      • Champoux J.J.
      • McConaughy B.L.
      ). After stirring for 40 min, the PEG precipitate was pelleted by centrifugation at 10,000 × g for 10 min. The resulting PEG supernatant was dialyzed against 4 liters of potassium phosphate buffer (PPB) (250 mM KPO4, pH 7.4, 1 mM DTT, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride). The dialyzed PEG supernatant was clarified by centrifugation at 10,000 × g for 10 min and passed over a 10-ml bed volume of phosphocellulose (Whatman P11) equilibrated with PPB. The P11 column was washed with 50 ml of PPB and then step-eluted with 30 ml of PPB containing 700 mM KPO4, pH 7.4. The P11 eluate was dialyzed overnight against 2 liters of PPB and passed over a 6-ml bed volume of phenyl-Sepharose (PS) (Pharmacia) that was equilibrated with PPB. The Topo I flowed through the PS column and was collected together with a 10-ml wash. An equal volume of water was then added to the PS flow-through, which was passed over a Mono Q (5H/R; Pharmacia Biotech Inc.) column that had been equilibrated with K100 buffer (100 mM KPO4, pH 7.4, 1 mM DTT, and 1 mM EDTA). The Topo I flowed through the Mono Q column and was loaded onto a Mono S column (5H/R) that was equilibrated with K100. The Mono S column was eluted with a 25-ml 50-200 mM KPO4, pH 7.4, gradient. Topo I eluted as a single major peak at ~150 mM KPO4. The peak Mono S fractions were pooled, concentrated with an Amicon ultrafiltration cell, dialyzed into storage buffer (50% glycerol, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA), and stored at −20°C. The high level expression achieved in the baculovirus-infected insect cell system yielded ~10 mg of protein from 3 × 109 cells.

       Purification of Recombinant Topo70 and Topo58

      The purification of recombinant Topo70 was as described above for the full-length Topo I. The initial steps in the purification of recombinant Topo58, up to and including elution of the P11 column, were as described for the full-length enzyme. The P11 eluate was then dialyzed against K100, filtered through a 0.45-μm syringe filter, and loaded onto a POROS SP20 (4.6/100) column (PerSeptive Biosystems) that was equilibrated with K100. The POROS SP20 column was eluted with a linear 100-500 mM KPO4, pH 7.4, gradient, and Topo58 was found to elute at 300 mM KPO4. The peak fractions were pooled, diluted with an equal volume of water, and loaded onto a Mono S column that was eluted with a 100-300 mM KPO4, pH 7.4, gradient. Approximately 70% of the Topo58 eluted from the Mono S at 200 mM KPO4, while the remainder eluted at 250 mM KPO4. The 200 mM peak fractions were pooled, diluted with an equal volume of water, and passed over a Mono Q column that was equilibrated with K100. The flow-through fractions were concentrated with an Amicon ultrafiltration cell, dialyzed into storage buffer and stored at −20°C.

       Purification of HeLa Topo I

      The starting material for purification of native human Topo I was 3 × 109 HeLa S3 cells that were doubling every 20-24 h in S-modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 50 units/ml of nystatin. The initial steps in purification of HeLa Topo I, up to the point of isolating the clarified dialyzed PEG supernatant, were identical to that described above for the recombinant enzyme. The dialyzed PEG supernatant was diluted with an equal volume of water, filtered through a 0.45-μm filter, and loaded onto a POROS SP20 (4.6/100) column that was equilibrated with cation exchange buffer (7 mM MES, 7 mM HEPES, 7 mM sodium acetate, pH 7.5) plus 100 mM NaCl. The SP20 column was eluted with a 30-ml linear salt gradient from 100 to 800 mM NaCl. Plasmid relaxation assays were performed to identify the peak Topo I fractions, which were found to elute at ~700 mM NaCl. The Topo I fractions were pooled, dialyzed against 2 liters of PPB, and passed over a 2-ml bed volume of PS that was equilibrated with PPB. The PS flow-through was diluted with an equal volume of water and loaded onto a Mono S column (5H/R) that was equilibrated with K100. The Mono S column was eluted as described for the recombinant enzyme, and the peak fractions were dialyzed into storage buffer and stored at −20°C.

       Purification of f-Topo70 and f-Topo75 Fragments of Recombinant Topo I

      During a 3-week room temperature incubation of the clarified PEG supernatant from a preparation of baculovirus-expressed Topo I, all of the full-length enzyme was proteolytically cleaved into a roughly equal mixture of 70- and 75-kDa forms of Topo I (designated f-Topo70 and f-Topo75, respectively). At this time, the PEG supernatant was clarified by centrifugation at 10,000 × g for 10 min and dialyzed against 4 liters of PPB. The dialyzed PEG supernatant was clarified by centrifugation at 10,000 × g for 10 min and loaded onto a POROS SP20 (4.6/100) column that was equilibrated with cation exchange buffer plus 400 mM NaCl. The proteins were eluted with a linear 50-ml NaCl gradient from 400 mM to 1 M. The f-Topo70 and f-Topo75 co-eluted at 500 mM NaCl. The peak SP20 fractions were pooled, dialyzed against 2 liters of K100, and loaded onto a Mono S column (5H/R) that was equilibrated with K100. The column was eluted with a 20-ml 100-200 mM KPO4, pH7.4, gradient. The f-Topo75 eluted at 140 mM KPO4, while the f-Topo70 eluted at 180 mM KPO4. The peak fractions of each protein were pooled, dialyzed against Mono Q Buffer (100 mM NaCl, 1 mM DTT, 1 mM EDTA, 20 mM Tris-hydrochloride, pH 7.5), and passed through a Mono Q column that was equilibrated with Mono Q buffer. Finally, the purified f-Topo70 and f-Topo75 fractions were dialyzed into storage buffer and maintained at −20°C.

       Amino-terminal Sequencing

      The f-Topo75 and f-Topo70 proteins were fractionated by SDS-PAGE and then transferred to Immobilon-P (Millipore) membranes in 10 mM CAPS, pH 10. The proteins were visualized by staining the membranes with Coomassie Blue. The appropriate bands were excised and sent to DNA Express (Fort Collins, CO) for amino-terminal sequencing.

       Gel Filtration and Glycerol Gradient Sedimentation

      Fast protein liquid chromatography (Pharmacia) gel filtration analyses were performed at 25°C with a flow rate of 0.75 ml/min using a Superose 12 (Pharmacia) column that was equilibrated with gel filtration buffer (200 mM KPO4, pH 7.4, 1 mM DTT, 1 mM EDTA). Purified protein samples of 10-100 μg were diluted into gel filtration buffer and injected in a total volume of 200 μl. Elution profiles of both Topo I constructs and molecular weight markers were monitored by UV absorbance at 280 nm and analyzed by SDS-PAGE. Glycerol gradient sedimentation analyses were performed by layering 250-μl samples of protein in 200 mM KPO4, pH 7.4, onto a 3.8-ml linear 10-30% glycerol gradient containing gel filtration buffer. The gradients were centrifuged at 50,000 rpm in an SW60 rotor (Beckman) for 16 h at 25°C. Fractions of 300 μl were collected from the bottom of the gradient tube through a small puncture and analyzed by SDS-PAGE and silver staining. The experimental gradients each included carbonic anhydrase as an internal standard. Parallel gradients were used to determine the sedimentation profiles of the marker proteins.

       Circular Dichroism

      Proteins were extensively dialyzed into 10 mM KPO4, pH 7.4. Exact molar concentrations of each protein were calculated from the A280 measurements of fully denatured protein in 6 M guanidine hydrochloride using molar extinction coefficients that were predicted by the Genetics Computing Group (GCG) software. CD spectra were obtained at room temperature, using a Jasco 3000 spectrapolarimeter with a 0.1-cm path length cell. The molar ellipticity spectrum for each sample was taken as the average of 8-12 individual scans. To eliminate the dichroism that is contributed by the sample buffer, the molar ellipticity spectrum for the dialysis buffer was subtracted from the molar ellipticity spectrum for each sample. The molar ellipticity values were normalized for the concentration of amide bonds in each sample and then converted to δϵ values(
      • Johnson C.W.J.
      ).

       SDS-Polyacrylamide Gel Electrophoresis and Autoradiography

      SDS-PAGE was performed according to Laemmli(
      • Laemmli U.K.
      ). Proteins were visualized by Coomassie Blue or silver staining(
      • Blum H.
      • Beier H.
      • Gross H.J.
      ). Autoradiography was performed by exposing dried gels to Kodak XAR film.

      RESULTS

       Expression and Purification of Recombinant Forms of Human Topo I

      With the long term goal of investigating the domain structure of human Topo I, we developed procedures to obtain large quantities of purified enzyme. This was achieved by the generation of recombinant baculoviruses that express wild type and active-site mutant (Y723F) versions of the full-length human Topo I and a 70-kDa protein (Topo70) which is missing the first 174 NH2-terminal amino acids (Fig. 2A). The recombinant Topo70 retains at least one (residues 192-198) of the four potential nuclear localization signals that reside in the NH2-terminal domain (
      • Alsner J.
      • Svejstrup J.Q.
      • Kjeldsen E.
      • S⊘rensen B.S.
      • Westergaard O.
      ) . We also generated a recombinant baculovirus that expresses a NH2- and COOH-terminally truncated 58-kDa protein (Topo58), which encompasses the conserved core domain (Fig. 2A). The recombinant proteins have been purified to apparent homogeneity (Fig. 2B). The wild type full-length Topo I and Topo70 proteins have the same specific activity as the native HeLa enzyme (Table 1, line 1). The active site Y723F mutant protein and Topo58 are at least 5000-fold less active than the wild type enzyme. The very low level of activity present in the mutant preparations is due to trace amounts of contaminating insect cell Topo I.
      Figure thumbnail gr2
      Figure 2:Recombinant proteins. Panel A, baculoviruses were engineered to express the following proteins: wild type and active-site mutant (Y723F) full-length human Topo I (F.L. topo I), wild type and Y723F mutant versions of a 70-kDa NH2-terminally truncated Topo I (Topo70), which initiates translation with an engineered methionine immediately upstream of Lys175, and an NH2- and COOH-terminally truncated 58-kDa form of Topo I (Topo58), which has the same initiating methionine as Topo70 but is terminated after residue Ala659. The predicted molecular mass (kDa) for each protein is indicated at the right. Panel B, purified proteins (5 μg each) were fractionated by 9-17% SDS-PAGE and visualized by Coomassie Blue staining. Lane 1 contained molecular mass markers (Bio-Rad) myosin (200 kDa), β-galactosidase (116 kDa), phosphoylase b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), lysozyme (14.4 kDa), and aprotinin (6.3 kDa). Lane 2, HeLa Topo I. Lane 3, recombinant Y723F full-length Topo I. Lane 4, Y723F proteolytic fragment of 75 kDa (f-Topo75). Lane 5, Y723F proteolytic fragment of 70 kDa (f-Topo70). Lane 6, Y723F recombinant Topo70. Lane 7, recombinant Topo58.
      Tabled 1
      Table thumbnail fxt1
      The recombinant full-length Topo I and HeLa cell Topo I co-migrate in SDS-PAGE analyses (Fig. 2B, compare lanes 2 and 3). Although the full-length proteins are predicted to be 91-kDa(
      • D'Arpa P.
      • Machlin P.S.
      • Ratrie H.I.
      • Rothfield N.F.
      • Cleveland D.W.
      • Earnshaw W.C.
      ), they migrate anomalously in SDS gels with an apparent molecular mass of ~100 kDa. In contrast, the recombinant Topo70 migrates appropriately with an apparent molecular mass of ~70 kDa (Fig. 2B, lane 6). Since it has previously been observed that negatively charged residues retard the migration of proteins in SDS gels(
      • Stewart L.
      • Vogt V.M.
      ), it is likely that the 48 negatively charged residues in the first 174 NH2-terminal amino acids are responsible for the slow migration of the full-length protein.
      While designing the purification scheme, we noticed that long term storage of nuclear extracts leads to proteolysis of the full-length enzyme into a fragments of 75 and 70 kDa, designated f-Topo75 and f-Topo70, respectively (Fig. 2B, lanes 4 and 5). Both of these fragments were purified and found to display activity equal to that of the full-length protein (Table 1, line 1), indicating that they retain the active site tyrosine, which is very close to the COOH terminus. This observation, together with the fact that amino acids downstream of the active site are essential for activity(
      • Kikuchi A.
      • Miyaike M.
      ),3 indicated that f-Topo75 and f-Topo70 represent NH2-terminally deleted forms of Topo I. Amino terminal sequencing confirmed that f-Topo75 was missing the first 137 residues while f-Topo70 was missing the first 174 residues.

       The Effect of Divalent Metal Cations and Polycations on Topo I Activity

      Although not required for Topo I activity, divalent metal cations are known to stimulate the activity as much as 25-fold (
      • Goto T.
      • Laipis P.
      • Wang J.C.
      ,
      • Liu L.F.
      • Miller K.G.
      ). Therefore to further characterize the recombinant full-length Topo I and Topo70, we examined the effect of a variety of divalent cations on the activity of the two proteins. In the standard relaxation assay buffer (which contains 150 mM KCl), Mg2+, Mn2+, Ba2+, and Ca2+ were all found to stimulate Topo I activity approximately 16-fold (Table 1, lines 2, 10, 19, 20, and 21). In contrast, Cd2+, Zn2+, Co2+, and Cu2+ completely inhibit Topo I (Table 1, lines 22-25), while Ni2+ was found to inhibit activity 16-fold at 5 mM (Table 1, line 26).
      To further investigate the stimulatory effect of Mg2+ we thought it would be informative to determine the level of Mg2+ stimulation obtained over a range of salt concentrations (Fig. 3). Initially, we varied the salt concentration in the absence of Mg2+ and found that the optimal KCl concentration was 200-250 mM, with very little activity detected at KCl concentrations less than 10 mM or greater than 400 mM (Fig. 3A, and data not shown). We then assayed the effect of 10 mM Mg2+ over a range of KCl concentrations from 25 to 350 mM. This revealed that Mg2+ had its largest stimulatory effect (50-fold) at low salt concentrations (25 mM KCl). As the KCl concentration was increased, the stimulatory effect of Mg2+ steadily dropped, and at ~250 mM Mg2+ was slightly inhibitory. With higher salt concentrations, 300 and 350 mM, the addition of Mg2+ was 3- and 7-fold inhibitory, respectively. The full-length and NH2-terminally truncated Topo70 enzymes behaved identically in their responsiveness to salt and Mg2+ (data not shown). For both forms of Topo I, there was an inverse relationship between the fold-effect of Mg2+ on activity and the KCl concentration. This is best depicted graphically as a logarithmic plot of the ratio of enzyme activity with and without Mg2+versus the KCl concentration (Fig. 3B).
      Figure thumbnail gr3
      Figure 3:The effect of Mg2+ and KCl on Topo I activity. Relaxation assays were initiated by mixing 10 ng of full-length Topo I (in 4 μl of dilution buffer 150 mM KCl, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA, 0.1 mg/ml BSA) with 130 μl of assay buffer (variable KCl concentration, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA, 0.1 mg/ml BSA, 0.025 μg/μl plasmid DNA, with or without 10 mM MgCl2) that had been prewarmed to 37°C. Reactions were incubated at 37°C, and at various time points 20-μl aliquots were stopped with SDS. The products were fractionated by 0.8% agarose gel electrophoresis and visualzed by ethidium bromide staining. Complete relaxation was said to have been achieved when supercoiled plasmid could no longer be detected visually. Panel A is a graphical representation of the log of the reciprocal of the time (min) required to fully relax the plasmid DNA versus the concentration of KCl. Panel B is a graphical representation of the log of the ratio (activity in the absence of Mg2+/activity in the presence of Mg2+) versus the concentration of KCl.
      Like divalent metal cations, polycations such as spermine and spermidine have also been shown to stimulate Topo I activity(
      • Srivenugopal K.S.
      • Morris D.R.
      ,
      • Shuman S.
      • Golder M.
      • Moss B.
      ). However, previous reports did not describe the effect of a combination of Mg2+ and a polycation, which might shed light on the stimulatory mechanism of each alone. For example, if the Mg2+ and spermidine effects were additive, then this might suggest separate mechanisms for activation. Thus, we examined the ability of spermidine and spermine to stimulate Topo I activity in the presence and absence of 10 mM MgCl2 (Table 1). In the absence of Mg2+, spermine (1 mM) and spermidine (5 mM) were found to stimulate Topo I activity 8-fold at 150 mM KCl. However, the combination of 10 mM Mg2+ and 1 mM spermidine stimulated the activity 64-fold. This suggests that the effects of polycations and Mg2+ are at least partially additive and that the two substances may stimulate Topo I activity by different mechanisms.

       Effect of ATP on Topo I Activity

      Topo I does not require ATP or any other energy source for activity. However, it has been reported that physiological concentrations of ATP (~2 mM) can inhibit human Topo I(
      • Castora F.J.
      • Kelly W.G.
      ). In another report, ATP was shown to inhibit human Topo I only in the presence of 1 mM KPO4(
      • Low R.L.
      • Holden J.A.
      ). These conflicting reports prompted us to examine the effect of ATP and KPO4 on the activity of HeLa Topo I in the presence or absence of 10 mM Mg2+ (Table 1). In the absence of Mg2+, 4 mM ATP had no detectable effect on Topo I activity whether 3 mM KPO4 was included or not (Table 1, lines 6 and 7). In the presence of 10 mM Mg2+, 4 mM ATP was found to inhibit the activity 2-fold regardless of the presence or absence of 3 mM KPO4 (Table 1, lines 8 and 9). Since ATP was only inhibitory in the presence of Mg2+, it seemed possible that the inhibitory effect of ATP could be the consequence of its ability to bind Mg2+, which would effectively lower the concentration of free Mg2+. To test this possibility, we assayed the activity in the presence of 6 mM Mg2+, which is the expected concentration of free Mg2+ in a mixture of 10 mM MgCl and 4 mM ATP. Activity in the presence of 6 mM Mg2+ was reduced 2-fold relative to activity in the presence of 10 mM Mg2+ (Table 1, compare lines 2 and 10). Similar results were obtained for both recombinant full-length Topo I and Topo70. Thus, we conclude that the inhibitory effect of ATP is not the result of binding to the enzyme but rather is the consequence of the ability of ATP to reduce the free Mg2+ concentration.

       The Effect of Camptothecin and Its Derivatives on Topo I Activity

      To further characterize the activity of the recombinant full-length Topo I and Topo70, we examined the inhibitory effects of camptothecin and its derivatives, topotecan and 9amino-camptothecin. When camptothecin was included in the reactions at 50 μM, activity was inhibited 8-16-fold in the presence or absence of 10 mM Mg2+, 1 mM spermine, or 5 mM spermidine (Table 1, lines 11-14). Topotecan and 9-amino-camptothecin were also found to inhibit relaxation 8-16-fold in the presence or absence of Mg2+ (Table 1, lines 15-18).

       Hydrodynamic Properties of Full-length Topo I and Topo70

      The sensitivity of the NH2-terminal one-fourth of Topo I to proteolysis suggested that it might be in an extended conformation, while the remaining three-quarters of the protein, which is more resistant to proteolysis, might be more globular. To test this notion, we subjected full-length Topo I, Topo70, and Topo58 to gel filtration and glycerol gradient sedimentation analyses (Fig. 4). As previously reported(
      • Kretzschmar M.
      • Meisterenst M.
      • Roeder R.G.
      ), human Topo I chromatographed through gel filtration with an apparent molecular mass of ~300 kDa (Fig. 4A). In contrast, the same protein sedimented in a glycerol gradient with an apparent molecular mass of ~66 kDa (Fig. 4B). The discrepancy in the molecular mass estimates by the two methods suggests that Topo I has an extended shape and thus a larger frictional coefficient than would be expected for a globular protein of 91 kDa. In contrast, the NH2-terminally truncated Topo70 was found to have an apparent molecular mass of ~66 kDa by sedimentation and ~96 kDa by gel filtration, indicating that the NH2-terminal region is largely responsible for the asymmetric shape of the protein.
      Figure thumbnail gr4
      Figure 4:Gel filtration and glycerol gradient sedimentation. Panel A, proteins were chromatographed over a Superose 12 column, and elution profiles were monitored by UV absorbance at 280 nm. Molecular mass standards (Sigma) were apoferritin (443 kDa), β-amylase (200 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anyhdrase (29 kDa), and lysozyme (14 kDa). Experimental samples were full-length recombinant Topo I (F.L. topo I), recombinant Topo70, and recombinant Topo58. The results are presented graphically as log (molecular mass) versus the ratio of observed elution volume (Ve) to excluded volume (Vo). The calculated apparent molecular masses for full-length Topo I, Topo70, and Topo58 are~300, ~96, and ~83 kDa, respectively. Panel B, proteins were fractionated by sedimentation through 10-30% glycerol gradients. Fractions were collected and analyzed by SDS-PAGE and silver staining. Molecular mass standards (Sigma) were β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anyhdrase (29 kDa). The results are presented graphically as molecular mass versus fraction number. The calculated apparent molecular masses for full-length Topo I, Topo70, and Topo58 are ~66, ~66, and ~60 kDa, respectively.
      Since full-length Topo I and Topo70 both co-sediment with BSA in a glycerol gradient, we can assume that the sedimentation coefficients for the two proteins are approximately equal to that of BSA (s020,w = 4.55)(
      • Freifelder D.
      ). Thus, the frictional coefficient (f) for each enzyme can be estimated from the formula f =m(1 - vρ)/s, (where m is molecular mass, v is the partial specific volume, ρ is the density of the solvent, and s is the sedimentation coefficient(
      • Freifelder D.
      ). The partial specific volumes for full-length Topo I and Topo70 are both ~0.74, as predicted from the amino acid content of each protein(
      • Freifelder D.
      ). Thus, taking ρ as equal to 1.0 g/cm3 (the density of water at 20°C), the frictional coefficients for the full-length protein and Topo70 are calculated to be 8.7 × 10-8 and 6.6 × 10-8 (g × s-1), respectively. Given these frictional coefficients, the axial ratio (
      • Freifelder D.
      ) of the full-length protein is approximately 10, as compared with a value of 5 for Topo70 (BSA is 6), confirming the elongated shape for full-length Topo I.
      Topo58 and Topo70 eluted from a gel filtration column with similar apparent molecular masses of ~83 and ~96 kDa, respectively. However they sedimented with apparent molecular masses of ~59 and ~66 kDa. The ~30-kDa disparity between molecular mass estimates by the two methods indicates that the shared 58 kDa domain of the two proteins either contains a small highly extended region or is by itself somewhat elongated. Evidence from limited proteolysis studies (see accompanying paper(
      • Stewart L.
      • Ireton G.
      • Champoux J.J.
      )) indicates that the former possibility is more likely to be correct. For example limited trypsin digestion of full-length Topo I generates a proteolytically resistant 55-kDa fragment that starts at residue Lys654 and ends somewhere very close to residue Lys654 (Fig. 1A in (
      • Stewart L.
      • Ireton G.
      • Champoux J.J.
      )). The sensitivity to proteolysis strongly suggests that the NH2-terminal segment of the Topo58 protein is likely extended (residues Lys174-Lys197), providing the hydrodynamic feature that leads to the ~30-kDa discrepancy.

       CD studies of Full-length Topo I and Topo70

      The gel filtration and sedimentation analyses indicated that human Topo I has a highly extended NH2 terminus, which minimally includes the first 174 residues but probably extends up to residue 197. To further investigate this structural feature, we obtained the far UV CD spectra for both the wild type and active-site mutant (Y723F) versions of full-length Topo I and Topo70 (Fig. 5). Within experimental error, the CD spectra for the wild type and mutant versions of the two proteins are identical (Fig. 5, A and B). Hence, as expected the active-site mutation Y723F does not appreciably alter the structure of the enzyme as measured in this way. In contrast, the averaged CD spectra of Topo70 and full-length Topo I are different (Fig. 5C). To minimize experimental error in this comparison, we averaged the mutant and wild type spectra obtained for two different preparations of each protein. Topo70 has a significantly stronger polarization in the 190-nm range than the full-length protein. Since polarization at 190 nm is a very reliable predictor of α-helical content in proteins(
      • Johnson C.W.J.
      ,
      • Johnson C.W.J.
      ), it is immediately apparent that the Topo70 protein has a greater percentage of α-helical content than the full-length protein. To confirm this notion, we compared the percentage secondary structures of the two proteins as predicted by the program VARSLC1(
      • Johnson C.W.J.
      ). The predicted molecular mass of α-helix (~28 kDa) and parallel β-sheet (~3 kDa) are very similar for the two proteins. However, the full-length protein is predicted to contain an extra 10 kDa of unfolded regions, 8 kDa of turns, and 4 kDa of antiparallel β-sheet. Hence, while the NH2 terminus appears to be largely unfolded, it also appears to have a substantial quantity of turns and antiparallel β-sheet. The turns and antiparallel β-sheet could either be folded into a stable contiguous domain or instead could be a reflection of transiently formed secondary structure in an otherwise random coil (
      • Johnson C.W.J.
      ). To assess which of the explanations is more likely, we analyzed the first 174 residues Topo I with the PEPTIDESTRUCTURE function of the GCG software package (Fig. 5D). This program predicts only very short regions of secondary structure for the NH2-terminal domain. Taking all of the information into account, gel filtration, sedimentation, and CD analyses, we conclude that the amino-terminal 174 resides of Topo I are largely unfolded and are comprised of very little if any extended secondary structure.
      Figure thumbnail gr5
      Figure 5:Circular dichroism. CD spectra are presented graphically as δϵ values versus wavelength (nm). Panel A shows representative spectra for wild type (solid line) and active site mutant (Y723F) (dashed line) versions of full-length Topo I. Panel B shows representative spectra for wild type (dashed line) and Y723F (solid line) versions of Topo70. Panel C depicts the average spectra for full-length Topo I (dashed line) and Topo70 (solid line), obtained by combining and then averaging mutant and wild type spectra (a total of seven spectra) for different preparations of each protein. Panel D is a graphical representation of the Chou-Fasman peptide structure prediction of residues 1-174 made by the GCG software. Sine waves, α-helix; sharp sawtooth, β-sheet; 180° change in direction, turns; dull sawtooth, random coil.

      DISCUSSION

       The Effects of Metal Cations and Polycations

      The recombinant human Topo I produced in the baculovirus-infected insect cell system displays the same apparent molecular weight and specific activity as the native enzyme purified from HeLa cells. Consistent with earlier findings(
      • Liu L.F.
      • Miller K.G.
      ,
      • D'Arpa P.
      • Machlin P.S.
      • Ratrie H.I.
      • Rothfield N.F.
      • Cleveland D.W.
      • Earnshaw W.C.
      ,
      • Kikuchi A.
      • Miyaike M.
      ), the full-length Topo I and the NH2-terminally truncated Topo70 display identical activities. Both enzymes are inhibited by camptothecin, topotecan, and 9-amino-camptothecin but not by ATP. The activities of both are stimulated by Mg2+, Ba2+, Ca2+, and Mn2+, but are strongly inhibited by Ni2+, Zn2+, Cu2+, Cd2+, and Co2+. The stimulatory effect of Mg2+ was found to increase with decreasing salt concentration. Under low salt conditions of (25 mM), the 50-fold stimulatory effect of Mg2+ resulted in a final level of activity that was still 10-fold less than that achieved with the most favorable condition, a combination of 10 mM Mg2+ and 200 mM KCl. This suggests that Mg2+ may stimulate the activity by two mechanisms, one similar to that achieved by monovalent cations alone and another that further stimulates activity in the presence of monovalent cations up to 200 mM KCl. At higher KCl concentrations (≥ 250 mM) Mg2+ was found to be slightly inhibitory, as has been observed for the rat liver and vaccinia Topo I enzymes(
      • McConaughy B.L.
      • Young L.S.
      • Champoux J.J.
      ,
      • Shuman S.
      • Golder M.
      • Moss B.
      ).
      There are several potential mechanisms whereby a divalent cation such as Mg2+ could effect a large stimulation of Topo I activity. For example Mg2+ is known to effectively shield the negative charge of the phosphate backbone of duplex DNA, which in addition to allowing the two strands to wind tighter (
      • Wang J.C.
      ) also reduces the effective diameter of the double helix(
      • Rybenkov V.V.
      • Cozzarelli N.R.
      • Vologodskii A.V.
      ,
      • Shaw S.Y.
      • Wang J.C.
      ), making it more favorable for two duplexes to lie on top of each other to form a node (
      • Bednar J.
      • Furrer P.
      • Stasiak A.
      • Dubochet J.
      • Egelman E.H.
      • Bates A.D.
      ). Since it has been shown that Topo I has a preference for binding to nodes(
      • Zechiedrich E.L.
      • Osheroff N.
      ,
      • Madden K.R.
      • Stewart L.
      • Champoux J.J.
      ), it could be envisioned that the presence of Mg2+-facilitated nodes recruits Topo I to supercoiled DNA, thereby effectively increasing activity. Alternatively, Topo I may simply prefer to relax DNA with a Mg2+-shielded phosphate backbone. Another possibility is that Mg2+ binds to the enzyme effecting some allosteric activation. However, the fact that Mg2+ does not influence the patterns of limited proteolysis of either the free or DNA-bound protein (see accompanying paper(
      • Stewart L.
      • Ireton G.
      • Champoux J.J.
      )), suggests that Mg2+ does not have any major effect on enzyme structure. For the vaccinia Topo I, Mg2+ has been shown to stimulate activity by accelerating the release of DNA substrate following topoisomerization, which is the rate-limiting step in steady state catalysis under low salt conditions(
      • Stivers J.T.
      • Shuman S.
      • Mildvan A.S.
      ). Since the viral enzyme has no detectable divalent metal cation binding site(
      • Stivers J.T.
      • Shuman S.
      • Mildvan A.S.
      ), this effect is presumably mediated by metal cation binding to DNA. Given the known effects of monovalent cations on processivity (
      • McConaughy B.L.
      • Young L.S.
      • Champoux J.J.
      ) and the similarity between the stimulatory effects of monovalent cations and Mg2+, it seems likely that the cellular enzymes are stimulated by Mg2+ in a manner similar to the viral enzyme. The possibility of direct participation of a divalent metal cation in phosphodiester bond cleavage would seem to be excluded by the fact that metal cations are not required for Topo I activity.
      The stimulatory effects of Mg2+ and the polycations (spermine and spermidine) were found to be at least partially additive, suggesting that the two substances may influence Topo I activity by separate mechanisms. Spermine and spermidine can effectively shield the phosphate backbone of duplex DNA by binding in the minor groove(
      • Schmid N.
      • Behr J.-P.
      ). This information and the fact that certain minor goove binding compounds are known to be inhibitors of Topo I (
      • Chen A.Y.
      • Yu C.
      • Gatto B.
      • Liu L.F.
      ) can be taken to suggest that the configuration of the minor groove can influence Topo I activity. This hypothesis is further supported by the observed weak consensus sequence for Topo I cleavage (5′-(A/T)(G/C)(A/T)T-3′), which is suggestive of protein contacts within the minor groove(
      • Been M.D.
      • Burgess R.R.
      • Champoux J.J.
      ,
      • Shen C.C.
      • Shen C.-K. J.
      ). Alternatively, spermine and/or spermidine may have an allosteric effect on Topo I. Further experiments are needed to better define the effects of both divalent and polyvalent cations on Topo I activity.

       The NH2-terminal Domain

      The role of the unconserved NH2-terminal domain remains elusive. All cellular eukaryotic topoisomerase I enzymes have this highly charged domain, which in every case examined is dispensable for in vitro activity(
      • Liu L.F.
      • Miller K.G.
      ,
      • Alsner J.
      • Svejstrup J.Q.
      • Kjeldsen E.
      • S⊘rensen B.S.
      • Westergaard O.
      ,
      • Kikuchi A.
      • Miyaike M.
      ,
      • Champoux J.J.
      • McConaughy B.L.
      ,
      • Bjornsti M.A.
      • Wang J.C.
      ). However, since this domain contains nuclear localization signals, it is nevertheless required for the in vivo function of Topo I(
      • Alsner J.
      • Svejstrup J.Q.
      • Kjeldsen E.
      • S⊘rensen B.S.
      • Westergaard O.
      ). Of the four putative nuclear localization signals residing in NH2-terminal domain of the human enzyme, residues Lys15-Asp156 have been suggested to be the most important for nuclear localization based on sequence comparisons with other cellular eukaryotic Topo I enzymes(
      • Alsner J.
      • Svejstrup J.Q.
      • Kjeldsen E.
      • S⊘rensen B.S.
      • Westergaard O.
      ). However, we find that Topo70, which starts at a methionine immediately 5′ to residue Lys1752, is transported to the nucleus of insect cells. Hence, the single remaining intact nuclear localization signal (Lys192-Glu198) must be sufficient for nuclear transport, at least in insect cells.
      Aside from its role in nuclear localization, what is the function of the NH2 terminus? In the human enzyme, we find that residues 1-174 of the NH2-terminal domain are largely if not completely unfolded and that the activity of the Topo70 enzyme (which is missing these residues) is indistinguishable from that of the full-length enzyme by every criterion tested. The highly extended nature of the NH2-terminal domain was observed using analytical techniques that involved conditions of both low salt (circular dichroism, 10 mM) and high salt (sedimentation and gel filtration, 200 mM). Hence, the extended nature of the NH2 terminus persists under variable salt concentrations and is not an artifact of any single analytical technique. With its high density of both negatively and positively charged residues (67% charged residues), the NH2 terminus is almost zwitterionic in nature. Accordingly, we have observed that the full-length enzyme can be concentrated to >50 mg/ml with no signs of precipitation. In contrast, Topo70 can only be concentrated to ~5 mg/ml before it begins to precipitate (data not shown). Hence, the NH2-terminal domain acts as a solubilizing element in vitro. A conservative estimate for the quantity of Topo I in HeLa cells can be made based on our recovery of 250 μg of purified Topo I from 109 HeLa cells. Since greater than 95% of the cell protein is associated with the nuclear compartment (data not shown), which has an average free water volume of approximately 3000 μM3 for HeLa S3 cells(
      • Rossini G.P.
      ), we estimate that the minimal nuclear concentration of Topo I is ~100 μg/ml. This concentration is 50-fold below the solubility limit of Topo70. However, Topo I is known to be highly concentrated in regions of chromatin that are undergoing high levels of transcription such as activated heat-shock loci and the nucleolus(
      • Baker S.D.
      • Wadkins R.D.
      • Stewart C.F.
      • Beck W.T.
      • Danks M.K.
      ,
      • Muller M.T.
      • Pfund W.P.
      • Mehta V.B.
      • Trask D.K.
      ,
      • Fleischmann G.
      • Pflugfelder G.
      • Steiner E.K.
      • Javaherian K.
      • Howard G.C.
      • Wang J.C.
      • Elgin S.C.R.
      ,
      • Rose K.M.
      • Szopa J.
      • Han F.S.
      • Cheng Y.C.
      • Richter A.
      • Scheer U.
      ). Furthermore, UV cross-linking and SDS-induced trapping of Topo I-DNA complexes has revealed that Topo I is enriched at least 20-fold on rDNA relative to total DNA (
      • Muller M.T.
      • Pfund W.P.
      • Mehta V.B.
      • Trask D.K.
      ) and is enriched 20-fold at induced heat shock loci relative to the uninduced loci(
      • Gilmour D.S.
      • Pflugfelder G.
      • Wang J.C.
      • Lis J.T.
      ). Thus, the possibility exists that local concentrations of Topo I could approach 5 mg/ml, which might necessitate a solubility factor such as the zwitterionic NH2 terminus.

       The Shape of Topo I

      In addition to revealing the extended nature of the NH2-terminal domain, the comparison of the hydrodynamic properties of full-length Topo I to those of Topo70 and Topo58 revealed that the core, linker, and COOH-terminal domains fold into a globular structure. Hence, the catalytically active domain of Topo I is largely globular, while the NH2-terminal domain is largely unfolded, consistent with it being dispensable for activity. Since the Topo58 core domain was capable of folding into a well behaved globular molecule, it probably represents a subdomain of Topo I that is capable of folding independent of the amino-terminal, linker, or core domains. Finally, the availability of milligram quantities of purified full-length Topo I, Topo70, and Topo58 greatly facilitated further studies of the structure-function relationships of Topo I. In the accompanying paper, we describe the use of limited proteolysis to further characterize the domain structure of the protein(
      • Stewart L.
      • Ireton G.
      • Champoux J.J.
      ).

      Acknowledgments

      We thank Sam Whiting, Sharon Schultz, Wim Hol, Xiayang Qiu, Lars Pedersen, and Dave Teller for helpful comments and valuable discussions during preparation of the manuscript. The POROS purifications were performed using the SPRINT high pressure liquid chromatography system from Perseptive Biosystems, kindly provided by Ken Walsh.

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