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Reconstitution of Enzymatic Activity by the Association of the Cap and Catalytic Domains of Human Topoisomerase I

2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês

10.1074/jbc.m205302200

1083-351X

Zheng Yang, James J. Champoux,

Bioactive Compounds and Antitumor Agents

When human topoisomerase I binds DNA, two opposing lobes in the enzyme, the cap region (amino acid, residues 175–433) and the catalytic domain (Δcap, residues 433 to the COOH terminus) clamp tightly around the DNA helix to form the precleavage complex. Although Δcap contains all of the residues known to be important for catalysis and binds DNA with an affinity similar to that of the intact enzyme, this fragment lacks catalytic activity. However, a mixture of Δcap and topo31 (residues 175–433) reconstitutes enzymatic activity as measured by plasmid DNA relaxation and suicide cleavage assays. Although the formation of an active complex between topo31 and Δcap is too unstable to be detected by pull-down experiments even in the presence of DNA, the association of topo31 with Δcap persists and is detectable after the complex catalyzes the covalent attachment of the DNA to Δcap by suicide cleavage. Removal of topo31 from Δcap-DNA after suicide cleavage reveals that, unlike the cleavage reaction, religation does not require the cap region of the protein. These results suggest that activation of the catalytic domain of the enzyme for cleavage requires both DNA binding and the presence of the cap region of the protein. When human topoisomerase I binds DNA, two opposing lobes in the enzyme, the cap region (amino acid, residues 175–433) and the catalytic domain (Δcap, residues 433 to the COOH terminus) clamp tightly around the DNA helix to form the precleavage complex. Although Δcap contains all of the residues known to be important for catalysis and binds DNA with an affinity similar to that of the intact enzyme, this fragment lacks catalytic activity. However, a mixture of Δcap and topo31 (residues 175–433) reconstitutes enzymatic activity as measured by plasmid DNA relaxation and suicide cleavage assays. Although the formation of an active complex between topo31 and Δcap is too unstable to be detected by pull-down experiments even in the presence of DNA, the association of topo31 with Δcap persists and is detectable after the complex catalyzes the covalent attachment of the DNA to Δcap by suicide cleavage. Removal of topo31 from Δcap-DNA after suicide cleavage reveals that, unlike the cleavage reaction, religation does not require the cap region of the protein. These results suggest that activation of the catalytic domain of the enzyme for cleavage requires both DNA binding and the presence of the cap region of the protein. glutathioneS-transferase NH2-terminal truncation of human topoisomerase I missing first 174 amino acids COOH-terminal truncation of topo70 missing last 106 amino acids human topoisomerase I from residues 175–320 human topoisomerase I from residues 175–433 NH2-terminal truncation of human topoisomerase I beginning at residue 433 dithiothreitol Eukaryotic type I topoisomerases promote the relaxation of supercoiled DNA by nicking and rejoining one of the strands of the DNA. These enzymes are important for many biological processes including DNA replication, transcription, and recombination (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2076) Google Scholar, 2Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2192) Google Scholar). Eukaryotic topoisomerase I, the poxviral topoisomerases, and some bacterial topoisomerases belong to the type IB subfamily of topoisomerases (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2076) Google Scholar,3Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar). The type IB subfamily members bind to double-stranded DNA and can relax either positive or negative supercoils in the absence of energy cofactors or divalent cations. DNA cleavage is initiated by the nucleophilic attack of the O-4 atom of the active site tyrosine on the scissile phosphate with the resultant covalent attachment of the enzyme to the 3′ end of the broken strand. Rotation of the DNA at the site of the break relaxes any supercoiling tension followed by religation of the DNA and release of the enzyme (2Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2192) Google Scholar, 4Gupta M. Fujimori A. Pommier Y. Biochim. Biophys. Acta. 1995; 1262: 1-14Crossref PubMed Scopus (284) Google Scholar). Human topoisomerase I is a member of the type IB subfamily and is composed of 765 amino acids (91 kDa). Sequence comparisons and limited proteolytic analyses in combination with crystallographic studies of the enzyme define four major domains: an NH2-terminal domain (Met1–Gly214), a core domain (Ile215–Ala635), a linker domain (Pro636–Lys712), and a COOH-terminal domain (Gln713–Phe765) (Fig.1A) (5Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1996; 271: 7602-7608Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar, 7Lue N. Sharma A. Mondragon A. Wang J.C. Structure. 1995; 3: 1315-1322Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The highly charged NH2-terminal domain is dispensable for enzymatic activityin vitro (5Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1996; 271: 7602-7608Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and contains nuclear targeting signals and binding sites for other proteins, such as nucleolin and SV40 large T antigen (8Bharti A.K. Olson M.O. Kufe D.W. Rubin E.H. J. Biol. Chem. 1996; 271: 1993-1997Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Simons D.T. Virology. 1996; 222: 365-374Crossref PubMed Scopus (76) Google Scholar, 10Champoux J.J. Prog. Nucleic Acid Res. Mol. Biol. 1998; 60: 111-132Crossref PubMed Google Scholar). Topo70 is a truncated form of human topoisomerase I with a molecular mass of 70 kDa that lacks residues 1–174 of the NH2-terminal domain but retains full enzyme activityin vitro (11Kikuchi A. Miyaike M. Anoh T. Ikeda H. Oguro M. Molecular Biology of DNA Topoisomerases and Its Application to Chemotherapy. CRC Press, Boca Raton, FL1993: 121-130Google Scholar, 12Stewart L. Ireton G.C. Parker L.H. Madden K.R. Champoux J.J. J. Biol. Chem. 1996; 271: 7593-7601Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The core domain is highly conserved and contains all of the residues directly implicated in catalysis except the active site Tyr723. The COOH-terminal domain is also highly conserved and contains the active site Tyr723. Separately purified COOH-terminal and core domains can interact with each other and reconstitute topoisomerase I activity in vitro (5Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1996; 271: 7602-7608Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13Stewart L. Ireton G.C. Champoux J.J. J. Mol. Biol. 1997; 269: 355-372Crossref PubMed Scopus (108) Google Scholar). The linker region that connects the core domain to the COOH-terminal domain is not conserved and is dispensable for activity in vitro, although without the linker the enzyme has a reduced processivity (12Stewart L. Ireton G.C. Parker L.H. Madden K.R. Champoux J.J. J. Biol. Chem. 1996; 271: 7593-7601Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In the crystal structure of the human topoisomerase I complexed with DNA, the protein clamps around the DNA with most of the protein-DNA contacts involving the core and COOH-terminal domains (6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar, 14Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Crossref PubMed Scopus (619) Google Scholar). The core domain can by further divided into three subdomains: 1) core subdomain I (residues 215–232, 320–433), 2) core subdomain II (residues 233–319), and 3) core subdomain III (residues 434–635). Core subdomains I and II form the top lobe or “cap” of the enzyme and cover the top of the DNA as the structure is usually oriented (6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar, 14Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Crossref PubMed Scopus (619) Google Scholar). Core subdomain II does not come in contact with the DNA in the structure, but the folding of core subdomain II is similar to that of the homeodomain region found in a family of DNA-binding proteins (6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar,14Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Crossref PubMed Scopus (619) Google Scholar). Two long positively charged α helices (α6 from core subdomain I and α5 from core subdomain II) form a “V”-shaped structure on the front end of the cap that may contact the DNA during the rotation process (6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar, 14Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Crossref PubMed Scopus (619) Google Scholar). Core subdomain III and the COOH-terminal domain form the bottom lobe of the protein. This region of the protein is homologous to the catalytic domain of the site-specific recombinases that include HP1 integrase, λ integrase, and Cre recombinase, and also to the catalytic domain of vaccinia topoisomerase (6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar, 15Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The bottom lobe of the protein is attached to the cap region through a long α helix extending upwards from core subdomain III on one side of the bound DNA and by a salt bridge on the other side of the DNA formed by amino acid side chains extending from a pair of loops in core subdomains I and III (6Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Crossref PubMed Scopus (776) Google Scholar, 14Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Crossref PubMed Scopus (619) Google Scholar). To dissect the function of the core domain and explore the functional relationship between the cap and the core subdomain III, we have studied the properties of three fragments of human topoisomerase I either alone or in pairwise combinations (Fig. 1A). Topo31 (residues 175–433), a 31-kDa fragment that consists of the cap region of the protein, binds DNA well, whereas topo17 (residues 175–320), a 17-kDa fragment that mainly comprises core subdomain II (the homeodomain-like region), cannot bind DNA. The third fragment corresponds to the catalytic domain of the enzyme and contains core subdomain III, the linker region, and the COOH-terminal domain (residues 433–765), and the fragment is referred to as Δcap. Although Δcap contains all of the elements required for catalysis and can bind DNA, it is catalytically inactive. However, when topo31 and Δcap are combined, they reconstitute enzymatic activity. All of the truncation mutants were generated using standard PCR methodology. To generate topo17, pGEX-topo70 DNA (13Stewart L. Ireton G.C. Champoux J.J. J. Mol. Biol. 1997; 269: 355-372Crossref PubMed Scopus (108) Google Scholar) was used as the PCR template, in combination with a plus-sense primer that starts at position 852 of the topoisomerase I cDNA sequence (16D'Arpa P. Machlin P.S. Ratrie III, H. Rothfield N.F. Cleveland D.W. Earnshaw W.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2543-2547Crossref PubMed Scopus (261) Google Scholar) and a minus-sense primer that contained two stop codons followed by an AvrII restriction site immediately after the codon for residue 320 of the protein. The PCR products were purified and digested with NdeI and AvrII and ligated to pGEX-topo70-WT DNA that had been cleaved with the same two restriction enzymes. To generate topo31, we used the same plus-sense primer and a minus primer that contains two stop codons followed by an AvrII restriction site after the codon for residue 433 of the protein. The PCR fragment was purified and digested with SphI and AvrII and ligated to the pGEX-topo70-WT that had been cut with the same two restriction enzymes. The Δcap mutant was generated using a plus-sense primer containing aBamH1 site followed by an AUG codon that annealed upstream of residue 433 and a minus-sense primer that annealed downstream of position 2392 of the topoisomerase I cDNA sequence. The PCR fragment was purified and digested with BamH1 and NheI and ligated to pFASTBAC1 topo70 (17Yang Z. Champoux J.J. J. Biol. Chem. 2001; 276: 677-685Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) that had been cut with the same two restriction enzymes. All of the mutations were confirmed by dideoxy sequencing. GST1-topo17 and GST-topo31 were expressed and purified from Escherichia coli TOP10F′ (Invitrogen) cells containing the pGEX-topo17 and pGEX-topo31 plasmids, respectively, as described previously for GST-topo12 (18Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1999; 274: 32950-32960Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The GST-topo17 and GST-topo31 fusion proteins were either stored bound to the glutathione-Sepharose 4B beads in 100 mm KCl, 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm DTT at 4 °C or removed from the beads with Factor Xa, followed by dialysis in storage buffer (50% glycerol, 10 mm Tris-HCl, pH 7.4, 2 mm DTT, 1 mm EDTA) and storage at −20 °C. Δcap was purified from the recombinant baculovirus-infected SF-9 insect cells as described previously for topo70 (12Stewart L. Ireton G.C. Parker L.H. Madden K.R. Champoux J.J. J. Biol. Chem. 1996; 271: 7593-7601Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Each reaction contained 500 ng of supercoiled pBluescript KSII(+) DNA and a 2-fold serial dilution of topo31 (1, 2, 4, 8, 16, 32, and 64 pmol) either with or without 4 pmol of Δcap in 20 μl of reaction buffer (100 mm KCl, 10 mm Tris-HCl, pH 7.4, 1 mm DTT, 1 mmEDTA). The reactions were incubated at 37 °C for 1 h and then stopped by the addition of 5 μl of 5× stop buffer (2.5% SDS, 25 mm EDTA, 25% Ficoll 40, 0.08% bromphenol blue, 0.08% xylene). The products were analyzed by electrophoresis in a 1% agarose gel, stained with ethidium bromide, and visualized with a UV illuminator. The duplex oligonucleotide suicide cleavage substrate CL14/CP25 was labeled and annealed as described previously (18Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1999; 274: 32950-32960Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The suicide cleavage reactions were carried out by incubating 2 μg of topo17, topo31, or Δcap alone or in the indicated combinations in 20 μl of reaction buffer with 5 ng of the suicide cleavage substrate at 23 °C for 3 h. Topo70 (0.5 μg) was used as a positive control for suicide cleavage. 5 μl of 5× SDS loading buffer (5% SDS, 20% glycerol, 100 mm Tris-HCl, pH 8.0, 5% 2-mercaptoethanol, 0.12% bromphenol blue) was added to quench the reactions. The samples were boiled for 5 min and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie Blue to visualize the protein bands and dried before exposure to film to detect the radiolabeled proteins. Covalent complexes used as substrates for the religation reaction were generated by suicide cleavage as described previously (18Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1999; 274: 32950-32960Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Suicide cleavage was carried out in 100 μl of reaction buffer containing 20 nm labeled suicide substrate and 0.5 μm topo70 for 1 h or 2.5 μm topo31 plus 2.5 μm Δcap for 6 h at 23 °C. The suicide cleavage reaction with topo70 was stopped by adding KCl to a final concentration of 0.5 m to prevent further cleavage during religation. High salt inactivation of the reaction with the reconstituted enzyme was unnecessary owing to the slow cleavage rate, and furthermore, high salt was found to dissociate the topo31-Δcap complex (see Fig. 7). The reactions were transferred to 37 °C and preincubated for 2 min. Religation was initiated by the addition of a 300-fold molar excess of the 11-mer religation acceptor oligonucleotide (R11) that is complementary to the region downstream of the cleavage site (18Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1999; 274: 32950-32960Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Aliquots of 10 μl were removed at different time points (5 s, 15 s, 30 s, 2 min, 5 min, and 60 min), and the reactions were stopped by the addition of an equal volume of 1% SDS. Religation was complete by the 60-min time point in both cases. Samples were ethanol-precipitated and dissolved in 10 μl of 1 mg/ml trypsin and digested at 37 °C for 1 h to remove all but a short topoisomerase-derived peptide from the covalent complexes. The samples were analyzed by electrophoresis in a 20% sequencing gel. The religation product migrates as a 23-mer and is well resolved from the oligonucleotide-peptide covalent complex that migrates slower than the uncleaved oligonucleotide. The percentage of religation at each time point was quantified using a phosphorimager and the ImageQuant software. The labeling and annealing of the 25-mer duplex oligonucleotide, CL25/CP25, has been described previously (18Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1999; 274: 32950-32960Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The DNA binding assay was carried out by incubating the labeled CL25/CP25 DNA (0.5 nm) with aliquots of 2-fold serial dilutions of the indicated protein in 10 μl of reaction buffer. For topo70, topo58, topo31, and Δcap, the protein concentrations used in the assay ranged from 1 μm to 8.7 nm. For topo17 and the lysozyme control, the concentrations extended from 10 μm to 87 nm. The reactions were incubated at 23 °C for 15 min before the addition of 2.5 μl of 50% glycerol, followed by analysis on a 6% native polyacrylamide gel at 4 °C. The running buffer contained 25 mm Tris-HCl, pH 8.5, and 162 mm glycine. Due to the high pI values for the topo70 protein and the truncation fragments used here (>9.0), free protein and protein-DNA complexes migrated to the cathode and therefore only the free oligonucleotides entered the gel. The amount of unbound oligonucleotide in the gel was quantified using a phosphorimager and the ImageQuant software. The dissociation constant (Kd) was estimated from the protein concentration at which one-half of the total duplex oligonucleotide was bound to the protein (19Carey J. Methods Enzymol. 1991; 208: 103-117Crossref PubMed Scopus (319) Google Scholar). The reaction was carried out by incubating GST-topo31 (∼2.5 μm on beads) with an equal molar concentration of Δcap in the presence of 20 nm labeled suicide substrate in 45 μl of reaction buffer. The reaction was rotated at 23 °C for 2.5 h to permit suicide cleavage, and then the reaction was divided into three equal portions. The first 15-μl portion was quenched by the addition of 5 μl of 5× SDS loading buffer and used as the control for the total amount of DNA-Δcap generated in the reaction by suicide cleavage. The second 15-μl portion was centrifuged at 10,000 rpm for 2 min to remove the beads, and the supernatant was added to 5 μl of 5× SDS loading buffer. The beads were washed once with 50 μl of reaction buffer and suspended in 20 μl of 1× SDS loading buffer, and both the supernatant and the beads were analyzed by SDS-PAGE to test for the association of Δcap-DNA with GST-topo31. The third 15-μl portion was centrifuged at 10,000 rpm for 2 min, and the beads were washed once with 50 μl of 1× reaction buffer, then suspended in 20 μl of 300 mm KCl, 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm DTT, and rotated at 23 °C for 5 min to elute the Δcap-DNA. The beads with the bound GST-topo31 were removed by centrifugation at 10,000 rpm for 2 min, and the supernatant containing the Δcap-DNA was adjusted to 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm DTT, and 100 mm KCl. Fresh glutathione-Sepharose 4B beads were added to the eluted Δcap-DNA and the mixture was rotated at 23 °C for 2.5 h to test whether Δcap-DNA associates nonspecifically with the beads. To test the effects of salt on the GST-topo31 interaction with Δcap-DNA, bead-bound covalent complexes were prepared as described previously and incubated for 5 min in 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm DTT, and KCl concentrations ranging from 100 to 400 mm. The beads were collected by centrifugation and resuspended in 15 μl of 1× SDS loading buffer and analyzed by SDS-PAGE. 5 μl of 5× SDS loading buffer was added to each supernatant and similarly analyzed. The gel was stained with Coomassie Blue and dried before exposure to film to compare the amount of labeled DNA present in the beads with the amount in the supernatant at the different salt concentrations. To generate the substrate for religation, GST-topo31 bound to beads (∼2.5 μm) was mixed with an equal molar amount of Δcap, and the mixture was incubated with 20 nm of labeled suicide substrate in reaction buffer in a total volume of 100 μl at 23 °C for 6 h to allow suicide cleavage to occur. The beads were collected by centrifugation, suspended in 100 μl of 300 mm KCl and 10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm DTT, and rotated at 23 °C for 5 min to elute the Δcap-DNA from the beads. The beads were removed by centrifugation at 10,000 rpm for 2 min. To eliminate all traces of GST-topo31, 5 μl of pre-equilibrated glutathione-Sepharose 4B beads were added to the supernatant and rotated at 23 °C for 30 min before removal of the beads by centrifugation. This step was repeated three times. The supernatant that contains the Δcap-DNA was divided into four 25-μl aliquots prior to preparing the samples for the religation assay. 50 μl of 10 mm Tris-HCl, pH 7.4, 1 mmEDTA, 1 mm DTT was added to two of the aliquots to make 75-μl reactions with a final KCl concentration of 100 mm. 3 μl of topo31 (0.7 μg/μl) was added to the first 75-μl reaction (+topo31), and an equal volume of storage buffer was added to the second 75-μl reaction (−topo31). The same procedure was used to prepare two additional reactions for religation except the KCl concentration was adjusted to 800 mm. All the reactions were incubated at 37 °C for 2 min before religation was initiated by the addition of 5 μl of the R11 religation oligonucleotide (final concentration, 2 μm). A 10-μl aliquot was removed prior to the addition of R11 for the zero time point analyses. 10-μl aliquots were removed at the indicated times and quenched by adding an equal volume of 1% SDS. All the reactions were ethanol-precipitated and digested with 10 μl of 1 mg/ml of trypsin at 37 °C for 1 h. The samples were analyzed on a 20% sequencing gel and subjected to phosphorimager analysis using ImageQuant software. The various fragments of human topoisomerase I employed in this study are shown schematically in Fig. 1, panel A. Both topo17 (residues 175–320) and topo31 have the same NH2 terminus as topo70, but topo17 extends to the end of core subdomain II, whereas topo31 (residues 175–433) includes all of the cap region of the protein. The Δcap fragment corresponding to the catalytic domain begins where topo31 ends and extends through to the COOH terminus of the protein. Fusion constructs of topo17 and topo31 containing NH2-terminal GST were expressed and purified from E. coli. The fusion proteins were either used directly or the GST portion was removed by Factor Xa prior to use. Topo58 (residues 175–659) and the Δcap fragment (residues 433–765) were purified from recombinant baculovirus-infected insect cells. Analysis of the purified proteins by SDS-PAGE (Fig. 1, panel B) showed that all four fragments were essentially homogenous. Although the Δcap fragment contains all of the amino acids that constitute the active site of the enzyme, no plasmid DNA relaxation activity was detectable in the purified fragment (Fig.2, lane 9). As expected, no relaxation activity was associated with topo31 alone (Fig. 2, lanes 2–8). However, relaxation activity could be reconstituted by the addition of topo31 to Δcap. Under these conditions (1-h incubation), activity was first detectable at approximately a 2:1 molar ratio of topo31 to Δcap, and essentially complete relaxation was achieved at a molar ratio of 4 (Fig. 2, lanes 13 and 14, respectively). A 5′ end-labeled suicide substrate that contained a 14-bp duplex with an 11 base 5′-tail (Fig. 3, top of panel B) was used to test the cleavage activity of the topoisomerase I fragments alone or in pairwise combinations. Upon cleavage and formation of the covalent complex with the 5′ end-labeled DNA, the AG dinucleotide at the 3′ end of the scissile strand is released, preventing religation. Suicide cleavage resulted in the formation of a labeled oligonucleotide-protein species that could be detected by SDS-PAGE analysis. The results showed that none of the protein fragments alone had cleavage activity and that combining topo17 with Δcap did not reconstitute cleavage activity (Fig. 3, lanes 2–5). However, the combination of topo31 with Δcap yielded a labeled protein band that migrated slightly above the Δcap protein band on the Coomassie Blue-stained SDS-polyacrylamide gel (Fig. 3, panels A and B, lane 6) and that corresponded in size to a Δcap-DNA covalent complex. The amount of cleavage observed for the combination of topo31 with Δcap was less than that observed with a smaller amount of topo70 (Fig. 3, lane 7). These results confirmed that topo31 and Δcap can reconstitute topoisomerase I cleavage activity. When a suicide cleavage time course combining topo31 and Δcap was performed, cleavage reached a plateau after ∼20 h of incubation under these conditions (data not shown). Religation was studied under single turnover conditions by assaying the ability of the covalent intermediate to attach a 5′-hydroxyl-terminated 11-mer to the cleaved oligonucleotide (12-mer) to form a 23-mer product (18Stewart L. Ireton G.C. Champoux J.J. J. Biol. Chem. 1999; 274: 32950-32960Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 20Stivers J.T. Shuman S. Mildvan A.S. Biochemistry. 1994; 33: 327-339Crossref PubMed Scopus (95) Google Scholar). The first step of the reaction was carried out by incubating the suicide cleavage substrate described previously with topo70 or with the reconstituted topo31-Δcap complex to generate the corresponding covalent complexes. The religation reactions were initiated by the addition of the 11-mer acceptor oligonucleotide to the reaction mixtures. The religation assay for topo70 was carried out at 0.5m KCl to promote dissociation of the topoisomerase after strand closure and to prevent recleavage of the product. Religation by the reconstituted enzyme had to be carried out at 100 mmKCl because higher salt concentrations dissociated the topo31-Δcap complex (see below). Under these conditions the cleavage rate by the reconstituted activity was too slow to interfere with the religation measurement. The samples were treated with trypsin to remove all but a short trypsin-resistant peptide from the topoisomerase I-DNA covalent complexes prior to analysis in a sequencing gel (Fig.4, panel A). The percentage of religated product formed at each time point was plotted for topo70 and the topo31-Δcap mixture (Fig. 4, panel B). The results indicated that the religation kinetics for the reconstituted topo31-Δcap complex and topo70 are very similar and that topo31 and Δcap can fully reconstitute the religation activity of topoisomerase I. Δcap contains all of the critical residues involved in catalysis yet it lacks enzymatic activity. This lack of catalytic activity could result from a reduced affinity of Δcap for DNA. To test this possibility a native gel mobility shift assay was used to measure the DNA binding properties of the various topoisomerase-derived fragments. Similar to topo70, topo31, topo17, and Δcap are positively charged, and because a covalently bound oligonucleotide only partially neutralizes the positive charge, the protein-DNA complexes fail to enter the native gel. Under these conditions, Kd is equal to the protein concentration at which the amount of unbound oligonucleotide observed in the gel has been reduced by a factor of 2 (19Carey J. Methods Enzymol. 1991; 208: 103-117Crossref PubMed Scopus (319) Google Scholar). Lysozyme has a similar pI value and was therefore used as a negative control for DNA binding by topo31, topo17, and Δcap. The binding assays showed that the affinity of the topo17 protein for the DNA was about the same as that of the lysozyme control, indicating that the binding is relatively nonspecific (Kd of ∼5 × 10−6m) (Fig. 5, inset). Topo31 bound DNA with a Kd of ∼4 × 10−7m, whereas Δcap bound the substrate DNA with a higher affinity (∼1 × 10−7m), which is only 2-fold lower than that of topo70 (∼5 × 10−8m). These results showed that topo31 binds DNA with a somewhat reduced affinity compared with topo70 and that the absence of activity for Δcap is not due to a failure to bind DNA. Topo31 and Δcap together can reconstitute complete topoisomerase I activity, indicating that topo31 interacts with and activates Δcap either before or after the addition of DNA. However GST-topo31 bound on the glutathione-Sepharose 4B beads failed to pull down a detectable quantity of Δcap after incubating the two proteins together either in the presence or absence of DNA (data not shown). This result could indicate either that the intera