Residues within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation*

Michael LisbyDagger, Jens R. Olesen, Camilla Skouboe, Berit O. Krogh§, Tobias Straub, Fritz Boege, Soundarapaudian Velmurugan||, Pia M. Martensen, Anni H. Andersen, Makkuni Jayaram||, Ole Westergaard, and Birgitta R. Knudsen**

From the Department of Molecular and Structural Biology, University of Aarhus, C.F. Møllers Allé, Building 130, DK-8000, Aarhus C, Denmark,  Medizinische Poliklinik, University of Würzburg Medical School, Klinikstrasse 6-8, Würzburg D-97070, Germany, and || Section of Molecular Genetics and Microbiology, University of Texas at Austin, Texas 78712

Received for publication, December 6, 2000, and in revised form, March 1, 2001

    ABSTRACT
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All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Eukaryotic topoisomerase I (topo I)1 is a monomeric enzyme that plays a major role in important cellular processes by regulating the topology of DNA. The enzyme relaxes negative and positive supercoils arising as a consequence of DNA processes such as DNA transcription, replication, recombination, and chromosome condensation (1).

Mechanistically, topo I acts by introducing transient single-strand breaks into the DNA double helix. The catalytic cycle can be subdivided into several steps including: (i) non-covalent DNA binding, (ii) cleavage, (iii) strand rotation, (iv) religation, and (v) enzyme turnover. The cleavage and religation events constitute two reverse phosphoryl transfer (transesterification) reactions. During the cleavage reaction, an active-site tyrosine residue of the enzyme is used as a nucleophile to break a phosphodiester bond of the DNA backbone, generating a covalent enzyme-(3'-phosphotyrosyl)-DNA linkage and a free 5'-hydroxyl group (2-4). This 5'-hydroxyl group provides the nucleophile for the religation reaction that restores intact DNA.

The solved crystal structure of an N-terminal-truncated version of the human topo I together with proteolytic analyses show that the enzyme is organized into four structural domains. These consist of an N-terminal domain (amino acids 1-206), a core domain (amino acids 207-635), a linker domain (amino acids 636-712), and a C-terminal domain (amino acids 713-765) (2, 3). The C-terminal domain contains the active-site tyrosine (Tyr723), which together with the catalytic residues Arg488, Lys532, Arg590, and His632 of the core domain constitutes the active site of the enzyme (4-8). Structural data show that the core and C-terminal domains form a clamp structure that wraps around the DNA and, together with the helix-turn-helix linker domain (1), contacts DNA in a region extending 4 base pairs upstream and 9 base pairs downstream of the cleavage site (3). Based on this structural information of the human topo I-DNA complex, a model for strand rotation (topoisomerization) has been proposed. According to this "controlled rotation" model, rotation of the cleaved strand around the intact strand is partially hindered by contacts between the rotating DNA and part of the core and linker domains (3). The involvement of the linker in controlling strand rotation has recently been supported by biochemical studies showing that the sensitivity of topo I toward camptothecin in relaxation depends on a functional linker domain (9). The recently published crystal structure of a topo I form encompassing a few residues of the N-terminal domain (amino acids 203-214) demonstrates an interaction between Trp205 and the hinge region of the core domain (37). This interaction has been proposed to be important for the flexibility of the enzyme clamp, allowing controlled strand rotation. Beside such speculations, the putative enzymatic function(s) of the N-terminal domain of topo I has remained unknown. Based on biochemical analyses, it is assumed to be largely unfolded or highly dynamic (2, 10). It is essential for nuclear localization of topo I, and four nuclear localization signals have been identified within the domain (11). For years it has been considered unimportant for catalysis, since the first purified forms of catalytically active topo I were in fact proteolytic degradation products lacking this domain (12, 13). The apparent dispensability of the N-terminal domain was recently supported by deletion studies showing that human topo I variants lacking either the first 174 (9) or the first 190 amino acids (14) show no obvious defects in vitro.

In the present work we have addressed the possible role of the N-terminal domain in topo I catalysis by comparing the in vitro activities of a truncated version of human topo I lacking amino acids 1-206 (p67) with those of the full-length enzyme (p91). We have found significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. The sum of our results held together with previous reports showing no effect of deleting the first 190 amino acids of the enzyme (14) suggests a particularly important role of amino acids 191-206 of the N-terminal domain in catalysis most probably by coordinating non-covalent enzyme-DNA interactions during the individual steps of topoisomerization.

    MATERIALS AND METHODS
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Reagents and Enzymes-- Me2SO (ACS grade), phenylmethylsulfonyl fluoride, cytochrome c, dApdG, and camptothecin were from Sigma. Camptothecin was dissolved in 50% (v/v) Me2SO at 600 µM and stored at -20 °C. [gamma -32P]ATP (7000 Ci/mmol) was from ICN. T4 polynucleotide kinase and bovine serum albumin were from New England Biolabs. pBR322 was from Roche Molecular Biochemicals. Glutathione-Sepharose columns, Source 15S, thrombin, and pGEX-2TK were from Amersham Pharmacia Biotech. Nitrocellulose filters were from Whatman.

Yeast Strains and Construction of Human TOP1 Expression Plasmids-- The Saccharomyces cerevisiae top1 null strain RS190 (15) was kindly provided by R. Sternglanz (State University of New York, Stony Brook, NY). Plasmid pHT143, for expression of recombinant full-length human topo I (p91) in S. cerevisiae, was described previously (16). Plasmid pHT148, for expression of a fusion of GST to the N terminus of amino acids 207-765 of human topo I (GST-p67), was constructed by first cloning a polymerase chain reaction fragment of pGEX-2TK containing the GST tag and the polylinker into a BamHI/EcoRI fragment of pHT143. The original BamHI site in pHT143 was destroyed, and the topo I gene was deleted from pHT143 by this cloning step, generating a new cloning vector referred to as pRS426-GAL-GST. Subsequently, a polymerase chain reaction fragment containing amino acids 207-765 of human topo I was cloned into a BamHI/EcoRI fragment of pRS426-GAL-GST, generating pHT148. pHT147, for expression of a GST-tagged N-terminal fragment (p25) of human topo I, was constructed by inserting a polymerase chain reaction fragment encoding amino acids 1-218 of topo I into the BamHI/EcoRI site of the pGEX-2TK vector. In the pHT148 and pHT147 constructs, the GST tag and the topo I fragments were separated by specific cleavage at a thrombin protease site.

Expression and Purification of Recombinant Forms of Human Topo I-- The plasmids pHT143 and pHT148 for expression of p91 and GST-p67, respectively, were transformed into the yeast S. cerevisiae strain RS190. Crude cell extracts from 12 liters of yeast culture expressing the p91 or GST-p67 were prepared, and the proteins were purified on two heparin-Sepharose columns and a phenyl-Sepharose column essentially as described previously (17). For purification of p67, the GST tag was cleaved off by thrombin before applying the protein preparations on the phenyl-Sepharose column. For comparability, p91 was subjected to this procedure too but leaving out the thrombin protease. The purified enzymes were concentrated, and the buffers were exchanged on a 0.5-ml Source 15S column. In the final step, p67 and p91 were eluted with a buffer containing 600 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 10% glycerol. A GST-tagged N-terminal fragment of human topo I, GST-p25, was expressed from the plasmid pHT147 in the bacterial strain BL21 (18). Bacteria were grown at 37 °C in 200 ml of LB medium containing 0.1 mg/ml ampicillin until A600 had reached 0.6. Expression of GST-p25 was induced by 1 mM isopropyl beta -D-thiogalactopyranoside for 1 h at 37 °C. Cells were harvested by centrifugation at 4,000 rpm for 10 min at 4 °C and resuspended in 10 ml of 1× PBS. Crude cell extract was obtained by sonication for 1 min on ice. Sonication was repeated 4-5 times separated by a 1-min incubation on ice, and cell debris was subsequently eliminated by centrifugation for 5 min at 14,000 rpm. The supernatant was loaded onto a 2-ml prepacked glutathione-Sepharose column by gravity flow, and the column was subsequently washed with 20 ml of ice-cold 1× PBS. Before eluting p25 in 500 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 10% glycerol, 15 units of thrombin protease was dissolved in 1.5 ml of 1× PBS and loaded onto the column, which was then sealed and incubated overnight at room temperature. Homogeneity of the p25 preparation was obtained on a 100-µl Source 15S column as described above for p67 and p100. An equal volume of 87% glycerol was added to the enzyme preparations before storage at -20 °C. Protein preparations were analyzed by SDS-polyacrylamide gel electrophoresis. After Coomassie staining, the intensities of the protein bands were compared with a dilution series of a bovine serum albumin standard to determine the protein concentrations.

Thrombin Cleavage-- In a standard digestion, 1 mg of GST-p67 was incubated with 150 units of thrombin for 1 h at room temperature in an 8-ml volume of 1× PBS containing 5 mM dithiothreitol. Subsequently, the digestion mixture was diluted to 50 ml with 1× PBS and passed over a 2-ml prepacked glutathione-Sepharose column three times to remove uncleaved fusion protein and liberated GST tag. The N-terminal amino acid sequence of p67 after thrombin cleavage was Gly-Ser-Arg-Arg-Ala-Ser-Val-Gly-Ser-Pro-Glu207-Glu208, with the start of the topo I sequence indicated in bold letters.

Synthetic DNA Substrates-- Oligonucleotides for construction of topo I suicide substrates were synthesized on a model 394 DNA synthesizer from Applied Biosystems. The sequences and the preparation of the two classes of suicide substrates containing a protruding noncleaved or cleaved strand were described in detail previously (16). The sequences for constructing the oligonucleotides for the non-suicide DNA fragment used for the filter binding assay were: OL100T, 5'-gctatacgaattcgctataattcatatgatagcggatccaaaaaagacttagaaaaaaaaaaagcttaagcaacatatggtatcgtcggaattcaatgag; OL100B, 5'-ctcattgaattccgacgataccatatgttgcttaagctttttttttttctaagtcttttttggatccgctatcatatgaattatagcgaattcgtatagc.

Topo I-mediated DNA Cleavage-- Standard cleavage reactions were performed by incubating 50 fmol of suicide DNA substrate (OL19/OL27) with 500 fmol of p91 or p67 in 20 µl of topo I reaction buffer (10 mM Tris-HCl, pH 7.5, 5 mM CaCl2, 5 mM MgCl2) at indicated temperatures. The cleavage reactions were stopped by the addition of NaCl to a final concentration of 375 mM, and cleavage products were analyzed on an 8% SDS Tris-glycine polyacrylamide gel.

Topo I-mediated Ligation-- Active topo I-DNA cleavage complexes containing the enzyme covalently attached at an internal (using DNA substrate OL19/OL27) or a terminal (using DNA substrate OL22/OL25) position were generated by preincubating 50 fmol of suicide DNA substrate with 500 fmol of topo I at 37 °C for 5 min. This reaction was performed as described for topo I-mediated cleavage and terminated by the addition of 300 mM NaCl. Intramolecular DNA ligation mediated by topo I covalently coupled at an internal position was performed by continuing incubation in the presence of 1 µM dApdG in 50 µl of topo I reaction buffer and 300 mM NaCl. Intermolecular DNA ligation mediated by topo I covalently attached at a terminal position was performed by incubating the cleavage complexes in the presence of 0.02 µM 28/28-mer duplex DNA (OL32/OL33) in 50 µl of topo I buffer and 300 mM NaCl. All reactions were stopped by the addition of SDS to a final concentration of 0.1% (w/v), and DNA was precipitated with 3 volumes of ethanol. The samples were subsequently digested with 1 mg/ml trypsin in 20 µl of 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA for 30 min at 37 °C. Finally, the samples were analyzed on 12% denaturing polyacrylamide gels.

Assays for Topo I-mediated Relaxation of Plasmid DNA-- The relaxation activity of human topo I was assayed for a series of enzyme concentrations and time points as indicated. Unless otherwise stated, reactions were performed in 20 µl of a standard relaxation buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 5 mM MgCl2) and 50 fmol of negatively supercoiled pBR322. Relaxation reactions were stopped by the addition of SDS to a final concentration of 0.1% (w/v). Samples were proteolytically digested with 0.5 µg/ml proteinase K for 30 min at 37 °C before separating the products on a 1% agarose gel with 0.5× Tris borate electrophoresis buffer at 50 V for 12 h at 4 °C. DNA was visualized by subsequent staining of the gel with 0.5 µg/ml ethidium bromide and the gel image was acquired using the Bio-Rad Gel Doc-2000 system.

DNA Binding Assay-- DNA binding of p25 and cytochrome c was assayed by nitrocellulose filter binding essentially as described previously (19, 20). The proteins were incubated with 20 fmol of 5'-end-labeled DNA substrate as indicated in a binding buffer containing 50 mM Tris-HCl, pH 7.5, 0.1 mM dithiothreitol, 5% glycerol, 1 mM EDTA, and 20 mM NaCl in a 50-µl reaction volume for 5 min at room temperature. Subsequently, the samples were applied to a 0.2-µm nitrocellulose filter under vacuum. To reduce nonspecific binding of the DNA substrate, the nitrocellulose filter was pre-wetted in binding buffer. After application of the samples, the filter was washed in 500 µl of binding buffer to remove unbound DNA substrate and dried. The percentage of input DNA retained on the filter was determined using PhosphorImager. The data were corrected for the nonspecific binding of free DNA, which was generally 1-2% of the total DNA.

Polyacrylamide Gel Electrophoresis-- Reaction products from the cleavage assays were mixed with 1/10 volume of an SDS sample buffer containing 10% glycerol (v/v), 4% SDS (w/v), 50 mM Tris-HCl, pH 6.8, 0.01% Serva Blue G (w/v), and 200 mM beta -mercaptoethanol. Next, the samples were applied to an 8% SDS Tris-glycine polyacrylamide gel (NOVEX) and were electrophoresed for 2 h at 100 V. Subsequent to electrophoresis, the gel was fixed for 15 min in 10% (v/v) acidic acid and dried.

The trypsin-digested products from the ligation assays were mixed with 1 volume of 80% (v/v) deionized formamide, 50 mM Tris borate, pH 8.3,, 1 mM EDTA, 0.05% (w/v) bromphenol blue, and 0.05% (w/v) xylene xyanol, and the mixtures were heated to 90 °C for 2 min and applied to a 12% denaturing polyacrylamide gel. The labeled reaction products were visualized by a model SF Molecular Dynamics PhosphorImager. The amount of topo I-mediated DNA cleavage and ligation was quantified by integrating the area under the curve for each radioactive band using the ImageQuant software from Molecular Dynamics.

    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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DNA Relaxation Activity of p67 and p91-- To investigate the effects on enzymatic activity of deleting the N-terminal domain of human topo I, recombinant p67 (amino acids 207-765) and p91 (amino acids 1-765) were purified from yeast (see Fig. 1) and assayed for DNA relaxation activity. Two sets of time course experiments were performed in a low salt buffer (5 mM MgCl2) at 37 °C. In the first set, with a 3-fold molar excess of plasmid DNA compared with enzyme, p67 relaxed supercoiled pBR322 16-64 times faster than p91 (Fig. 2A). In the second set, with the enzyme concentration increased to a 2-fold molar excess compared with plasmid substrate, the relaxation rate of p67 was slower than that of p91 (compare lanes 2 and 8 in Fig. 2B). Thus, the relative activity of p67 with respect to p91 is dependent on the molar ratio between enzyme and DNA.


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Fig. 1.   Purification of p67 and p91 forms of topo I. A, domain organization of p67 and p91. B, SDS-polyacrylamide gel electrophoresis analysis of 2 µg of purified p67 and p91 forms of human topo I stained with Coomassie Blue.


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Fig. 2.   Comparison of DNA relaxation activity of p67 and p91. A, relaxation of supercoiled pBR322 by p67 and p91 in a molar excess of DNA. Lanes 1 and 7, 50 fmol of pBR322. Lanes 2-6, 50 fmol of pBR322 incubated with 17 fmol of p67 at 37 °C for 0.3, 1, 4, 16, and 64 min, respectively. Lanes 8-12, same as lanes 2-6 using p91 instead of p67. B, DNA relaxation by p67 and p91 in a molar excess of enzyme. Lanes 1 and 7, 50 fmol of pBR322. Lanes 2-6, 50 fmol of pBR322 incubated with 100 fmol of p67 at 37 °C for 10, 20, 40, 80, and 160 s, respectively. Lanes 8-12, as lanes 2-6 except that p67 was replaced with p91. SC, negatively supercoiled pBR322. RL, relaxed pBR322.

At a molar excess of plasmid DNA, the rate-limiting step for relaxation of the total substrate population is the enzyme-DNA dissociation rate (21). When changing the molar ratio between enzyme and DNA to an excess of enzyme, the majority of DNA molecules become occupied by at least one enzyme molecule. In consequence, enzyme-DNA dissociation is no longer rate-limiting for relaxation. In this context, our results suggest that deleting the N-terminal domain of human topo I increases the dissociation rate of the enzyme to yield a higher turnover number. This interpretation is further supported by the more distributive relaxation mode of p67 relative to p91 (Fig. 2A, compare lanes 2-4 with lanes 8-10), where the truncated enzyme appears to leave its substrate after removing only a few supercoils at a time. At the conditions used, the relaxation mode of full-length topo I is known to be highly processive, going through multiple rounds of relaxation before dissociating from its substrate (22, 23). Our data with p91 are consistent with this expectation. The relatively low relaxation activity of p67 at a molar excess of enzyme (Fig. 2B) may be explained in two ways. Either it reflects a slower catalysis of p67 relatively to p91 or a lower DNA affinity of p67 and a consequent reduction in the percentage of DNA-bound enzyme at a given time.

Involvement of the Human Topo I N-terminal Domain in DNA Binding-- The shift from a processive to a more distributive relaxation mode of topo I upon removal of amino acids 1-206 suggests a role of the N-terminal domain (or at least part of the N-terminal domain) in non-covalent DNA binding. To evaluate this possibility, we compared the salt sensitivity of p67 and p91 in relaxation and used this as a measure for their respective DNA affinities. It is well known that high salt concentration inhibits topo I-catalyzed DNA relaxation by inhibiting DNA binding (24).

The two enzymes were incubated with supercoiled pBR322 at various concentrations of NaCl, and the resulting products were analyzed by gel electrophoresis. The amount of supercoiled pBR322 remaining after incubation with topo I was quantified, and the relaxation activity was plotted as a function of the salt concentration (Fig. 3A). The p67 topo I showed a significantly lower salt optimum (25-75 mM NaCl) than p91 and was completely impaired in relaxation at 150 mM of NaCl. By contrast, the relaxation optimum for p91 was in the 100-150 mM NaCl range. These results are consistent with a lowered DNA affinity of p67. In agreement with this finding, an N-terminal fragment of human topo I (amino acids 1-218) binds DNA in a filter binding assay (Fig. 3B). Taken together, the obtained results suggest a role of the N-terminal domain or at least part of it in non-covalent DNA binding.


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Fig. 3.   DNA binding properties of p25, p67 and p91. A, salt optimum for DNA relaxation mediated by p67 and p91. 50 fmol of pBR322 was reacted with 5 fmol of either p67 or p91 in the presence of indicated NaCl concentrations for 1 h at 37 °C, and the resulting products were analyzed on a 1% agarose gel. Subsequent to ethidium bromide staining a digital image of the gel was acquired. The amount of supercoiled pBR322 remaining at each salt concentration was quantified and divided by the amount of supercoiled pBR322 in the control reaction with no enzyme to obtain a measure for relaxation activity, which is depicted graphically as a function of salt concentration. B, DNA binding of p25. Binding of p25 to double-stranded DNA was tested by incubation with 20 fmol of a 100-base pair fragment of 5'-end labeled duplex DNA (OL100T/OL100B) for 5 min at room temperature. Enzyme-bound DNA was separated from unbound DNA by filtration through a nitrocellulose membrane, which retains specifically protein-DNA complexes. Cytochrome c (C) was used to demonstrate that a non-DNA-binding protein with an overall charge similar to that of p25 fails to retain radiolabeled duplex DNA on the nitrocellulose filter. The resulting filter of such an experiment is shown in the upper panel. The amount of radiolabeled DNA retained on the filter was quantified on a PhosphorImager, normalized to the total amount of radiolabel, and plotted as a function of the protein concentration (lower panel). Filled diamonds, p25. Filled circles, cytochrome c.

The N-terminal Domain Is Important for Interaction with DNA Downstream of the Cleavage Site-- For further characterization of the N-terminal domain in the individual chemical steps of topo I catalysis, the cleavage and ligation reactions of p67 and p91 were compared using synthetic suicide DNA substrates (16, 25, 26). These substrates support cleavage, whereas religation is temporarily prevented due to dissociation of a short oligonucleotide containing the 5'-OH end generated during cleavage (Fig. 4A, right panel). Cleavage complexes containing the enzyme covalently attached at an internal or a terminal position can be obtained by using substrates with strand interruptions located next to the cleavage site on either the scissile or the non-scissile strand, respectively (16). DNA ligation mediated by the active cleavage complexes can be initiated by the addition of an excess of appropriate ligator strands containing free 5'-hydroxyl ends. Cleavage complexes containing the enzyme attached at an internal position are able to ligate single-stranded DNA complementary to the base sequence of the noncleaved strand (referred to as intramolecular ligation; Fig. 4B, right panel). Intramolecular ligation can be performed with ligators as short as 2 bases and proceeds in the presence of 1 M NaCl. Thus, non-covalent DNA interaction is not required for intramolecular ligation (27). Intermolecular ligation, i.e. ligation of duplex DNA to cleavage complexes carrying topo I covalently attached at a blunt end (Fig. 4C, right panel), is completely abolished at 1 M salt and depends strongly on non-covalent enzyme interactions to the ligator strands.


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Fig. 4.   DNA cleavage and ligation rates at 37 °C. The cleavage and ligation assays are schematically depicted in the right panels. The left panels are graphic representations of the obtained results. A, DNA cleavage rate for p67 and p91. A radiolabeled suicide DNA substrate was incubated with the topo I variants for the indicated time periods. Cleavage products were analyzed by SDS-polyacrylamide gel electrophoresis, and the percentage of cleaved DNA substrate was plotted as a function of incubation time. B, rate of intramolecular DNA ligation by p67 and p91. Active cleavage complexes containing p67 or p91 attached at an internal position were reacted with 1 µM dApdG. The reactions were stopped at the indicated times by the addition of SDS, and the reaction products were analyzed by denaturing gel electrophoresis. The amount of cleavage complexes converted to ligation product in each sample was plotted as a function of time. C, rate of intermolecular DNA ligation by p67 and p91. Performed as in panel B except that cleavage complexes containing p67 or p91 attached at a blunt end were reacted with a 28-mer duplex DNA. Filled triangles, p67. Filled squares, p91. Asterisk, 5'-radiolabeling. Filled circles, 5'-phosphorylation. T, topo I.

The cleavage reactions of p67 and p91 were investigated by incubating the enzymes with the suicide substrate OL19/OL27 at 37 °C. Fig. 4A shows the extent of covalent complex formation plotted as a function of time. The cleavage reactions for both enzymes were quite fast, completed within the first 40 s of incubation, and no significant difference between the two enzymes could be observed at these conditions. Note, however, that at low temperatures, the cleavage rates were markedly different for p67 and p91 (see below; Fig. 7A).

To investigate intra- and intermolecular ligation, topo I was mixed with the appropriate suicide DNA substrate to obtain active cleavage complexes with the enzyme covalently attached at an internal (substrate OL19/OL27) or a terminal position (substrate OL22/OL25), respectively. After incubation for 5 min at 37 °C, the salt concentration was increased to 300 mM to prevent further cleavage, and the appropriate ligator strands were added (5'-HO-dApdG for intramolecular ligation and OL32/OL33 for intermolecular ligation). Figs. 4, B and C, show the percentage of cleavage complexes converted to ligation products plotted as a function of the incubation time. As shown in Fig. 4C, the ability of p67 to mediate intermolecular ligation was severely compromised compared with p91, whereas the effect of deleting the N-terminal domain on intramolecular ligation was quite modest (no more than a 2-fold reduction in the initial reaction rate of p67 compared with p91; Fig. 4B). From the data in Fig. 4C, we estimate that the initial reaction rate for intermolecular ligation is at least 25-fold slower for p67 than for p91.

The most obvious difference between the two forms of ligation is their different requirements for enzyme interaction to DNA downstream of the cleavage site. During intramolecular ligation, the ligator strand can be positioned by complementary base pairing, so that the 5'-OH group may be oriented within the topo I active site for nucleophilic attack on the phosphotyrosyl bond. Due to the absence of base pairing, intermolecular ligation depends on protein-mediated positioning of the ligator strand. Accordingly, the ligation results suggest that residues within the N-terminal domain promote enzyme interaction to DNA downstream of the cleavage site. Such a function would be consistent with the lowered DNA affinity of p67 relative to p91 (Fig. 3A) and with the ability of the N-terminal domain to bind DNA non-covalently (Fig. 3B).

Camptothecin Sensitivity of p67 and p91 in Relaxation-- The structural and biochemical data on human topo I have led to the proposal of the controlled rotation model to explain the topoisomerization step of catalysis. According to this model, strand rotation is controlled by enzyme-DNA contacts downstream of the cleavage site (1, 3). The multiple contacts of the linker domain (amino acids 636-712) to DNA downstream of the cleavage site (1, 3) together with the recent finding that blockage of strand rotation by camptothecin depends on an intact linker (9) suggest that this domain could play a role in controlling topoisomerization. In this study we present evidence that residues of N-terminal domain, like the linker region, are important for enzyme interaction to DNA downstream of the cleavage site either by direct interaction(s) or by coordinating DNA interaction(s) of other parts of the enzyme. This would suggest an expanded version of the controlled rotation model in which residues of the N-terminal domain along with the linker domain are involved in controlling strand rotation. To address this possibility, the effect of deleting the N-terminal domain on the camptothecin sensitivity of the enzyme was investigated.

The camptothecin sensitivities of p67 and p91 in relaxation were assayed in molar excess of enzyme over DNA to circumvent possible effects due to a slow dissociation rate and enzyme turnover in the presence of camptothecin. Note that camptothecin blocks religation and will thereby slow down all the subsequent steps of topo I catalysis. However, first it was necessary to slow down the reaction rate, which is too fast under standard conditions employing 5 mM MgCl2 at 37 °C (see Fig. 2B), for the inhibition by camptothecin to be measured accurately. We found that excluding MgCl2 from the reaction buffer slows down the reactions of both p67 and p91 sufficiently (compare Figs. 5 and 2) (10, 26, 28-31). To obtain more physiologically relevant conditions, 75 mM NaCl, at which concentration the two enzymes show similar activities (see Fig. 3), was added to the reaction mixture. Thus, the effect of camptothecin (60 µM) was assayed at 37 °C in the absence of MgCl2 and presence of 75 mM NaCl (Fig. 5). The relaxation rate of p67 was practically unaffected by camptothecin (Fig. 5A, compare lanes 7-12 with lanes 1-6), whereas that of p91 was decreased ~16-fold by the drug (Fig. 5B, compare lanes 7-12 with lanes 1-6). The drug resistance of p67 in relaxation is not due to a lowered binding affinity of the drug since camptothecin was found to be equally competent in stabilizing cleavage complexes introduced by p67 or p91 on double-stranded DNA fragments (data not shown). Rather, the N-terminal domain is likely required for camptothecin to block DNA strand rotation. Truncated versions of human topo I consisting of amino acids 175-765 or 191-765 have previously been reported to retain camptothecin sensitivity in relaxation (9, 10, 14). Thus, taken together the available data indicate that the peptide region spanning amino acids 191-206 is important for drug sensitivity in relaxation, which in turn implicates this region in the controlling of strand rotation.


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Fig. 5.   Sensitivity of p67 and p91 to camptothecin. Relaxation of supercoiled pBR322 by p67 and p91 assayed in the absence or presence of 60 µM camptothecin (CPT). A, sensitivity of p67 to camptothecin. Samples containing 50 fmol of pBR322 and 100 fmol of p67 were incubated at 37 °C for indicated periods of time. Lanes 1 and 7, 50 fmol of pBR322. Lanes 2-6, samples stopped after 0.3, 1, 4, 16, and 64 min of incubation with p67 in the presence of 5% Me2SO. Lanes 8-12, same as lanes 2-6 but incubated in the presence of 60 µM camptothecin and 5% Me2SO. B, sensitivity of p91 to camptothecin. Same as panel A using p91 instead of p67. SC, negatively supercoiled pBR322. RL, relaxed pBR322.

Temperature Sensitivity of p67-- While optimizing conditions for assaying the camptothecin effect (Fig. 5), we discovered a striking difference between p91 and p67 in their sensitivity to low temperatures (Fig. 6). The two enzyme forms were roughly equally active at 37 °C. However, at 0 °C the relaxation rate of p91 dropped ~64-fold relative to that at 37 °C (compare lanes 22-24 of Fig. 6A with lanes 10-12 of Fig. 6B), whereas the corresponding activity drop for p67 was much higher, leaving the enzyme almost inert in relaxation (Fig. 6A, lanes 2-12). At 45 °C, the relaxation activities of p67 and p91 were comparable (data not shown), as was the case at 37 °C.


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Fig. 6.   Temperature dependence of DNA relaxation mediated by p67 and p91. Relaxation of supercoiled pBR322 by p67 and p91 at different temperatures. A, DNA relaxation at 0 °C. The reaction mixture was prepared and incubated on ice. Samples were withdrawn at the indicated times and stopped by the addition of SDS. Each sample contained 50 fmol of pBR322 and 100 fmol of either p67 or p91. Lanes 1 and 13, 50 fmol of pBR322. Lanes 2-12, samples were stopped after 0.2, 0.3, 0.7, 1.3, 2.7, 5.3, 11, 21, 43, 85, and 171 min of incubation with p67. Lanes 14-24, same as lanes 2-13 but using p91 instead of p67. B, DNA relaxation at 37 °C. The reaction mixture was prepared and incubated at 37 °C, but otherwise the treatments were as in panel A. Lanes 2-6, samples were stopped after 0.2, 0.3, 0.7, 1.3, and 2.7 min of incubation with p67. Lanes 8-12, same as lanes 2-6 but with p91 replacing p67. SC, negatively supercoiled pBR322. RL, relaxed pBR322.

The cold sensitivity of p67 could be due to a conformational change, leaving the enzyme completely inactive at 0 °C, or any single one of the catalytic steps could be defective. To investigate these possibilities, the cleavage and ligation reactions were analyzed separately at 0 °C. Fig. 7A shows that the initial rate of cleavage by p67 at 0 °C was decreased ~11-fold relative to p91, whereas there was no significant difference between the ligation rates of p67 and p91 at 0 °C (Fig. 7B). These experiments show that p67 is capable of catalyzing transesterification at 0 °C, indicating that the active site of the enzyme is competent in transesterification chemistry even at low temperatures. The reduced ability of p67 to mediate strand cleavage may therefore rather be due to defects in the catalytic steps before cleavage such as DNA binding and/or in bringing the enzyme-DNA complex into a proper conformation for cleavage.


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Fig. 7.   DNA cleavage and ligation rates at 0 °C. Cleavage and ligation rates were assayed essentially as described in the legend for Fig. 4, but with the modification that all reactions were incubated on ice. A, DNA cleavage rates for p67 and p91 at 0 °C. The percentage of cleaved DNA substrate is plotted as a function of incubation time. B, rates of intramolecular DNA ligation by p67 and p91 at 0 °C. The amount of cleavage complexes converted to ligation product in each sample is plotted as a function of time. Filled triangles, p67. Filled squares, p91. Asterisk, 5'-radiolabeling. Filled circles, 5'-phosphorylation. T, topo I.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although divergent in amino acid sequence, the N-terminal domain has been preserved through evolution as a highly basic region in all cellular type IB topoisomerases (32). In human topo I, a number of nuclear localization sequences, phosphorylation sites, and protein interaction sites have been mapped to the N-terminal domain (11, 32-36). These diverse features are suggestive of multiple modes of topo I regulation in vivo. However, the N-terminal domain has long been thought to contribute little to the topo I enzyme activity per se. Here we describe for the first time a significant modulation of in vitro DNA relaxation mediated by residues within this domain.

The salient findings from our present work may be briefly summarized as follows. In the absence of the N-terminal domain, relaxation by human topo I becomes more distributive. The p67 enzyme is unaffected in intramolecular ligation but severely depressed in intermolecular ligation. DNA relaxation by p67 is unimpeded by camptothecin, whereas p91 is inhibited ~16-fold by the drug. The p67 protein also differs from p91 in its cold sensitivity in both DNA cleavage and relaxation, whereas intramolecular strand ligation mediated by p67 is not subject to this temperature effect. As explained below, all of these observations can be accommodated by a model according to which residues within the N-terminal domain participate in the physicochemical steps of topoisomerization by mediating enzyme-DNA contacts either by binding DNA directly or by promoting DNA binding of other regions of the enzyme.

The role of N-terminal domain in DNA binding, initially suggested by the distributive relaxation mode of p67, was further supported by nitrocellulose binding assays using double-stranded DNA targets, which demonstrated an inherent DNA binding affinity of this domain. The data are most consistent with the potential interaction site of DNA downstream of the cleavage site. Two pieces of evidence argue in favor of this interpretation: the contrasting effects of the N-terminal domain in intramolecular versus intermolecular ligation as well as the ability of p67 to carry out relaxation unimpeded by camptothecin. A similar effect on camptothecin sensitivity was recently reported for a form of human topo I lacking the linker domain (amino acids 636-712) (9). Under the assumption that camptothecin inhibits DNA relaxation by blocking a putative controlled strand rotation step of catalysis, the interpretation of this result was that the linker domain participates in controlling rotation of the cleaved strand during topoisomerization. Under the same assumption, our results suggest that the N-terminal domain could also be important for controlling strand rotation. Thus, based on the available data, we propose that residues from both the N-terminal and the linker domain may act together to control this step of catalysis. Two N-terminal-truncated versions of human topo I encompassing amino acids 175-765 or 191-765 (9, 14) have previously been shown to retain camptothecin sensitivity in relaxation. In combination with our results, the region spanning residues 191 through 206 is therefore highlighted as particularly important for catalysis. Interestingly, the recently published structure of human topo I including amino acids 203-214 shows the proximal part of the N-terminal domain to be located close to the cleavage site, supporting that this area of the enzyme could interact with the rotating strand (37). Moreover, circumstantial evidence for our general conclusions on DNA binding is provided by the presence of 13 lysine residues in the region between residues 175 and 202. In principle, the involvement of the N-terminal domain in catalysis is well substantiated alone by its apparent ability to contact DNA downstream of the cleavage site. However, the reduced DNA affinity of p67 could also, completely or in part, result from the lack of Trp205, which has recently been shown to interact with a hinge region within the core domain (37). This interaction is believed to be important in transition between an open and a closed clamp conformation of topo I upon DNA binding. A possible reduced capacity for this conformational change in the absence of the N-terminal domain would probably result in an overall decrease in DNA binding affinity and a reduced ability to coordinate DNA contacts during the individual steps catalysis.

The initially surprising observation that the p67 enzyme is almost inert in relaxation and strongly impaired in cleavage at 0 °C while retaining normal capacity for intramolecular DNA ligation is, in retrospect, not inconsistent with our interpretations. The cold sensitivity likely reflects the inability of p67 to induce conformational changes within itself and/or the DNA at low temperatures. Two non-exclusive explanations for the cold sensitivity of p67 can be hypothesized. One plausible explanation is that p67 is less flexible than the full-length enzyme due to the lack of interaction between the hinge region and Trp205, as discussed above. At low temperatures, the lack of such interaction may restrain transition of the enzyme from an open to a closed clamp conformation and thereby reduce the DNA binding affinity. Alternatively or in combination with the above, the p67 enzyme could be defective in mediating conformation changes in the DNA substrate, e.g. bending or partial unwinding of the DNA helix, which may be inherent to the process of cleavage and topoisomerization. A similar phenomenon has recently been observed for two cold-sensitive mutants of the DnaA protein from Escherichia coli (38). These mutants are defective in initiating DNA replication at 20 °C due to their inability to unwind the DNA double helix. At higher temperatures the free energy of the DNA is sufficient to overcome the defect of the mutants.

The in vitro functions of the N-terminal domain of human topo I suggested by the present study may have important implications for the in vivo activity of the enzyme. It has been shown that topo I can be regulated via phosphorylation of residues located in the N-terminal domain (32, 33, 39), which may in fact change the DNA binding properties of this domain. In addition the relaxation activity of the enzyme is regulated by a number of proteins: SV40 large T-antigen, p53, casein kinase II, PSF, nucleolin, non-histone HMG proteins, and histone H1, all of which interact with and stimulate topo I activity (34, 40-44). All interaction domains mapped so far involve the N-terminal domain of topo I (34, 40, 43), and consequently, the interactions may interfere with the DNA binding properties of this domain. In support of the N-terminal domain importance for modulating topo I activity in vivo, band depletion experiments using camptothecin have shown that p67 is strongly impaired in cleavage in vivo,2 suggesting a role of the N-terminal domain for interaction with DNA assembled into chromatin.

    ACKNOWLEDGEMENTS

We are grateful to Kirsten Andersen and Inger Bjørndal for skillful technical assistance.

    FOOTNOTES

Dagger Present address: Dept. of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 West 168th St., New York, NY 10032.

§ Present address: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021.

** To whom correspondence should be addressed. Tel.: 45-89422703; Fax: 45-89422612; E-mail: brk@mbio.aau.dk.

Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M010991200

This work was supported by Danish Cancer Society Grants 9710032, 9910012, and 9910013, the Alfred Benzon Foundation, the Danish Research Councils, the Biotechnological Research Program (Biotec III), and the Danish Center for Molecular Gerontology. Support for the Boege laboratory was from Deutsche Forschungsgemeinschaft Grants SFB 172/B12, Bo 910/2-1, and Bo 910/3-1. Support for the Jayaram laboratory was provided by the Robert F. Welch Foundation, The Texas Board for Coordinating Higher Education, and the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2 M. O. Christensen and C. Mielke, personal communication.

    ABBREVIATIONS

The abbreviations used are: topo I, topoisomerase I; p91, human topo I (amino acids 1-765); p67, human topo I (amino acids 207-765); p25, human topo I (amino acids 1-218); GST, glutathione S-transferase; PBS, phosphate-buffered saline; dApdG, 2'-deoxyadenylyl(3' right-arrow 5')-2'-deoxyguanosine.

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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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