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Originally published In Press as doi:10.1074/jbc.M309692200 on May 11, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30073-30080, July 16, 2004
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Thermotoga maritima-Escherichia coli Chimeric Topoisomerases

ANSWERS ABOUT INVOLVEMENT OF THE CARBOXYL-TERMINAL DOMAIN IN DNA TOPOISOMERASE I-MEDIATED CATALYSIS*

Thierry Viard{ddagger}, Raynald Cossard, Michel Duguet, and Claire Bouthier de La Tour§

From the Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Batiment 400, Université Paris Sud, Centre d'Orsay, 91405 Orsay Cedex, France

Received for publication, September 2, 2003 , and in revised form, May 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial topoisomerases I are generally composed of two domains as follows: a core domain, which contains all the conserved motifs involved in the trans-esterification reactions, and a carboxyl-terminal domain, highly variable in size and sequence. In the present work, we have addressed the question of the respective roles of the two domains in the different steps of the topoisomerization cycle. For this purpose, we prepared various recombinant topoisomerases from two model enzymes: topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima and topoisomerase I from Escherichia coli. We compared the properties of the two core domains to that of the topoisomerases formed by combining the core domain of one enzyme to the carboxyl-terminal domain of the other. We found that, contrary to E. coli (Lima, C. D., Wang, J. C., and Mondragon, A. (1993) J. Mol. Biol. 232, 1213–1216), the core domain from T. maritima (TmTop65) is able to sustain by itself a complete topoisomerization cycle, although with low efficiency. Fusion of TmTop65 to the entire carboxyl-terminal domain from E. coli considerably increases binding efficiency, thermal stability, and DNA relaxation activity. Moreover, the chimera predominantly acquires the cleavage specificity of E. coli full-length topoisomerase. For the chimera obtained by fusion of the T. maritima carboxyl-terminal domain to the core EcTop67, very low DNA relaxation activity and binding are recovered, but formation of a covalent DNA adduct is impaired. Taken together, our results show that the presence and the nature of the carboxyl-terminal domain of bacterial topoisomerases I strongly determine their DNA binding efficiency and cleavage specificity but is not strictly required for strand passage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The control of DNA topology is critical for all essential processes that require opening of the double helix such as DNA replication, transcription, DNA repair, or recombination. In hyperthermophilic organisms, this control appears more critical, as high temperature also helps open the double-stranded DNA. Thus, topoisomerases as they are able to modify DNA structure (for reviews, see Refs. 2 and 3) appear essential in these processes of adaptation to extreme temperatures.

We have recently described the properties of the topoisomerase I from the hyperthermophilic bacterium, Thermotoga maritima, whose growth temperature is about 80 °C (4). At 75 °C, this enzyme has an extremely high rate of relaxation activity on negatively supercoiled DNA. It belongs to the large family of topoisomerases IA present in all three domains of life, Bacteria, Archaea, and Eukarya (5, 6). This type of enzymes catalyzes the transient breakage of one strand in double-stranded DNA to allow passage of the other DNA strand, or duplex, through the break and then religation of the broken strand. The different steps of the topoisomerization cycle occur through trans-esterification reactions in which the active site tyrosine of the enzyme becomes linked to the 5'-phosphate.

An important advance in the understanding of the mechanism of topoisomerases IA has been made by the determination of the spatial structures of the Escherichia coli topoisomerase III (7) and the 67-kDa fragment polypeptide from E. coli topoisomerase I (1, 8, 9). This fragment, called Top67, contains the first 596 amino acids of the protein. It constitutes the core domain of E. coli topoisomerase I in which are present the totality of the conserved motifs, particularly those forming the active site of the protein (5, 10). The structural data combined with biochemical approaches have allowed us to postulate an enzyme-bridged mechanism for topoisomerases IA (11–14).

Like E. coli topoisomerase I, the enzyme from T. maritima can be divided into the following two domains: a core domain consisting of the first 540 amino acids of the protein, and a carboxyl-terminal domain consisting of the 93 remaining amino acids (Fig. 1).



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  		FIG. 1.
A, schematic diagram of the full-length, truncated, and chimeric topoisomerases I from T. maritima and E. coli. Sequences from T. maritima correspond to the black parts and those of E. coli correspond to the gray parts. TmTopo I represents the full-length T. maritima topoisomerase I (633 amino acids) and EcTopo I the full-length E. coli topoisomerase I (864 amino acids). The truncated topoisomerases TmTop65 and EcTop67 correspond to the 540 and 596 first amino acids of TmTopo I and EcTopo I, respectively. Long (LC) and short (SC) chimera result in the addition of the 268 or 157 last amino acids of EcTopo I to TmTop65, respectively. Reverse chimera (RC) results from the addition of the last 93 amino acids of TmTopo I to EcTop67. Sequence conserved motifs are represented by white boxes. Y* represents the tyrosine of the active site. B, amino acid sequences in the region of proteins where fusions have been made. Sequences belonging to T. maritima are black framed and sequences belonging to E. coli are gray framed. To construct the LC, SC, and RC chimera, two supplementary amino acids (one valine and one aspartic acid, underlined) have been added to the junction of both topoisomerases.

 
Whereas the core domains of bacterial topoisomerases I exhibit a high sequence similarity, the carboxyl-terminal domains are very divergent (5, 10, 15). They contain a variable number of zinc domains ranging from 0 to 5 (4, 16). In E. coli topoisomerase I, there is a large carboxyl-terminal domain (268 amino acids) with three tetracysteine zinc-binding motifs (17). Zinc deletion inactivates the enzyme (18), and mutation of one cysteine of the second zinc finger abolishes the cleavage specificity (19). Moreover, the core domain Top67 is inefficient in DNA relaxation and can cleave only small oligonucleotides (1). Recently, a potential role of the zinc domain in the strand passage step has been proposed (20).

In T. maritima topoisomerase I, the role of the 93 last amino acids constituting the carboxyl-terminal domain appears different from that of the E. coli enzyme. Indeed, we have shown that the hyperthermophilic enzyme contains a unique zinc motif that is not essential to the DNA relaxation activity and whose mutation does not change the intensity or specificity of DNA cleavage (4).

In an effort to better understand the respective roles of the carboxyl-terminal and core domains of the T. maritima topoisomerase I compared with those of E. coli, we have constructed, overexpressed, and purified eight recombinant proteins. These include the two full-length topoisomerases I, an inactive mutant of T. maritima enzyme, the two core domains, TmTop65 and EcTop67, and three chimeric proteins with the core domain of one protein fused to the carboxyl terminus of the other, and vice versa (Fig. 1). The different proteins were tested for various properties as follows: zinc content, ability to relax DNA, thermal stability, DNA binding, cleavage efficiency, and specificity. The results of these experiments provide a new insight into the respective roles of the core and carboxyl-terminal domains in the mechanism of bacterial topoisomerases I.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Protein Expression—The pET29b expression vector was used for all constructions. Recombinant T. maritima topoisomerase I (TmTopo I)1 was obtained as described previously (4).

A DNA fragment encoding the 540 first amino acids from T. maritima topoisomerase I was prepared by PCR, using genomic DNA as template, Pfu DNA polymerase (Expand high fidelity kit, Roche Applied Science), and the following primers: 5'-AGAGGTGGAGGAGGATCCGAGTAAGAAAGTGAAGAAATATATCGT-3' as the forward primer, containing a BamHI restriction site (underlined sequence), and 5'-GTCGTTCCTGTCGTCGACTCAGGAAAAGGATTCATAAAACTC-3' as the reverse primer, containing a SalI restriction site. PCR products were inserted into the pET29b expression vector (Novagen) at the BamHI/SalI sites, and constructs were checked by DNA sequencing. This construction was named TmTop65. When the carboxyl-terminal part of E. coli topoisomerase I was fused to TmTop65 to yield a chimeric protein, the stop codon was removed from the TmTop65 construct.

To allow the construction of chimeric proteins, DNA constructs encoding full-length protein and polypeptides from E. coli topoisomerase I were prepared. E. coli topoisomerase I (EcTopo I) was obtained by PCR from genomic DNA with 5'-AAATTAGGTAAGGTGCATATGGGTAAAGCTCTTGTC-3' as the forward primer, containing an NdeI restriction site, and 5'-ACCTGACAGAATGTCGACTTATTTTTTTCCTTCAAC-3' as the reverse primer, containing a SalI restriction site. PCR products were inserted into the pET29b expression vector at the NdeI/SalI sites.

A DNA fragment encoding the first 596 amino acids from E. coli topoisomerase I, also known as "Top67 mutant" (1), was also prepared with 5'-AGAAGGAGATATACATATGGGTAAAGCTCTTGTC-3' as the forward primer, containing an NdeI restriction site, and 5'-AGTCGGGCAGTCGACCTAGCTGGTCAGAACCATCTGGTT-3' as the reverse primer, containing a SalI restriction site. PCR products were inserted at the NdeI/SalI sites of pET29b. This construct was renamed EcTop67. When the carboxyl-terminal domain of T. maritima topoisomerase I was fused to EcTop67 to yield the chimeric protein RC, the codon stop was removed from the EcTop67 construct.

Two other DNA fragments encoding the last 268 and 157 amino acids from E. coli topoisomerase I were produced by PCR using as forward primers 5'-GTTCTGACCAGCGTCGACATTGACTGCCCG-3' and 5'-GGTTATGACGGCGTCGACGTTGAGTGTGAA-3', respectively, each containing a SalI restriction site. For both constructs, the oligonucleotide 5'-AAAACCTGACAGAGCGGCCGCTTATTTTTTTCC-3' was used as the reverse primer containing a NotI restriction site. These fragments were then cloned at the end of TmTop65 at the SalI/NotI sites. The chimeric proteins resulting from these constructions were named long chimera (LC, when the fragment corresponding to the 268 amino acids from EcTopo I was fused) and short chimera (SC, when the fragment corresponding to the 157 amino acids of EcTopo I was fused).

A DNA fragment encoding the last 93 amino acids from TmTopo I was obtained by using the two oligonucleotides: 5'-TATGAATCCGTCGACAGTGTGTTCGACAGGAAC-3' as the forward primer containing a SalI restriction site, and 5'-TGGTGCTCGAGTGCGGCCGCAAGCTTGTCCACTCAAGAGCCTTT-3' as the reverse primer, containing a NotI restriction site. This fragment was cloned at the end of EcTop67 DNA construct. The chimeric protein was named reverse chimera (RC). As a result of the cloning procedures, the chimeric proteins LC, SC, and RC contained two supplementary amino acids, one valine and one aspartic acid.

All the resulting expression plasmids were transformed into E. coli Rosetta (DE3) (Novagen). A colony of transformed cells was picked and grown at 37 °C in LB medium with 35 µg/ml kanamycin and 35 µg/ml chloramphenicol until the A600 reached 0.5. Induction of gene expression was carried out with 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside for 4 h at 37 °C(A600, 1.0–1.2), and the cells were harvested and stored at –80 °C until required. About 15 g of cells were obtained from 4.8 liters of culture. The same procedure was followed to express all the recombinant proteins.

Protein Purification—Frozen cells were resuspended in 10 volumes of buffer A (50 mM Tris-HCl, pH 7.5, 0.1 M NaCl), lysed by sonication, and centrifuged at 40,000 x g for 15 min at 4 °C. To remove DNA, Polymin P was added to the supernatant to a final concentration of 0.36% (w/v). After stirring for 1 h, the solution was centrifuged for 1 h at 90,000 x g. Ammonium sulfate (70% saturation, final concentration) was added to the supernatant, and the solution was allowed to stir overnight at 4 °C before centrifugation (24,000 x g for 30 min). The pellet was dissolved in 7 ml of buffer A and extensively dialyzed against this buffer at 4 °C. The solution was clarified by centrifugation at 24,000 x g for 20 min and applied onto an heparin-Sepharose column (IBF) equilibrated with buffer A. After washing with buffer A and buffer B (50 mM Tris-HCl, pH 7.5, 0.3 M NaCl), the elution was performed by applying a gradient from buffer B to a buffer consisting of 50 mM Tris-HCl, pH 7.5, 1.1 M NaCl. Active fractions eluted around 0.5–0.6 M NaCl. They were pooled and concentrated in an Amicon concentrator (Centricon 30) before loading onto a gel filtration column (Sephacryl S-200 HR, Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 7.5, 0.6 M NaCl. Glycerol was added to a final concentration of 15% (v/v), prior to storage of the active fractions at –20 °C. Protein concentrations were determined by the method of Schaffner and Weissmann (21) with BSA as a standard or spectrophotometrically by using the theoretical molar extinction coefficient of each recombinant protein.

Zinc Content Measurements—The zinc content of the wild type and mutant proteins was determined by a colorimetric method. A solution of 2.5 mM p-[hydroxymercuri]benzene sulfonate (PMPS) was added in increments of 1 µl to a 500-µl sample of a 10 µM enzyme solution (in 10 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5% glycerol) containing 0.1 mM 4-[2-pyridylazo]resorcinol (PAR). Zinc release from the enzyme was monitored by the absorbance at 500 nm of the PAR2 Zn(II) complex, whose molar absorptivity is 6.6 104 M–1 cm–1 (22).

DNA Relaxation Assay—The reaction mixture (10 µl) contained 50 mM Tris, pH 8.0, 0.5 mM DTT, 30 µg/ml BSA, 10 mM MgCl2, 120 mM NaCl, and 200 ng of negatively supercoiled plasmid DNA (pTZ 18). The reaction was initiated by adding the protein solution to be tested, which was serially diluted in a buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM DTT, 100 µg/ml BSA. The undiluted protein solution added to the reaction mixture contained 1 pmol of enzyme. After 30 min of incubation at 37 °C, the reaction was stopped by adding 2 µl of stop solution containing 50 mM EDTA, 2.5% SDS, 25% glycerol, and 0.2% bromphenol blue. Samples were electrophoresed through 2.5% agarose gels in TEP buffer (90 mM Tris phosphate, pH 8.0, 2 mM EDTA) for 5 h at 4 V/cm. Gels were stained with 1 µg/ml ethidium bromide and photographed under UV light.

Thermostability Assay—The proteins were preincubated at 50 °C for different times prior to dilution and tested for their DNA relaxation activity as described above.

Oligonucleotide Radiolabeling—The 22-mer oligonucleotide 5'-GAATGAGTCGCAACTTC{downarrow}GGGAT-3', containing a unique cleavage site for the T. maritima topoisomerase I (position indicated by the arrow) (4), was synthesized and purified by MWG Biotec prior to labeling at its 5'- or 3'-end. Ten pmol of this 22-mer oligonucleotide were 5'-end-labeled using 5 units of T4 polynucleotide kinase (Biolabs) in the presence of 20 µCi of [{gamma}-32P]ATP in 10 µl of buffer as recommended by the manufacturer. After 30 min of incubation at 37 °C, the reaction was stopped by heating the samples at 75 °C for 15 min. This labeling allowed us to evaluate the ability of topoisomerase I to interact with the noncovalently bound end of the cleaved oligonucleotide. Indeed, in the trans-esterification mechanism of type IA topoisomerases, the tyrosine of the active site forms a covalent intermediate with the 5'-end of the cleaved strand, leaving free the 3'-end which nevertheless remains complexed to the protein. The oligonucleotide size markers (Amersham Biosciences) were labeled following the manufacturer's protocol. For 3'-labeling, 20 pmol of the 22-mer oligonucleotide were incubated with 10 units of terminal deoxynucleotidyltransferase (Amersham Biosciences) in the presence of 50 µCi of [{alpha}-32P]ddATP in 50 µl of buffer as recommended by the manufacturer. After 1 h of incubation at 37 °C, the reaction was stopped by heating the samples to 95 °C for 5 min.

Gel Mobility Shift Assay and Oligonucleotide Cleavage Assay—0.2 pmol of the 5'-end-labeled oligonucleotide was incubated with 4 pmol of protein in buffer A, containing 50 mM Tris-HCl, pH 8.0, 0.5 mM DTT, 30 µg/ml BSA, and 60 mM NaCl for 30 min at 37 °C in a final volume of 20 µl. The reaction mixture was then split as follows: 10 µl were added to 2.5 µl of a buffer containing 10 mM Tris, pH 7.5, 20% glycerol, 1 mM EDTA, 0.1 mg/ml BSA, 1 mg/ml xylene cyanol; and 5 µl were loaded onto a 6% native polyacrylamide gel containing 50 mM Tris, 0.1 mM EDTA, 8 mM glycine. Electrophoresis was carried out at 4 °C at 150 V for 3 h and subjected to autoradiography. Quantitation was performed using the ImageQuant version 1.2 software. The second half of the reaction mixture was stopped by the addition of 1% SDS and 10 µl of loading buffer (97.5% formamide, 10 mM EDTA, 0.3% bromphenol blue, 0.3% xylene cyanol). The samples were denatured by heating at 95 °C for 3 min, and 8 µl were loaded onto an 18% denaturing polyacrylamide gel (19:1) containing 50% (w/v) urea in TBE buffer (90 mM Tris, 90 mM boric acid, 1 mM EDTA). The gel was electrophoresed at 48 V/cm for 2 h and subjected to autoradiography. Quantitation was performed using the ImageQuant version 1.2 software.

In some experiments (Fig. 7), the 20-µl reaction mixture was loaded onto a 12% native polyacrylamide gel instead of a 6% gel; 10 µl were treated as described above, and the remaining mixture was further incubated with 1% SDS and 0.5 mg/ml proteinase K at 50 °C for 45 min prior to being loaded onto the gel.



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  		FIG. 7.
Comparison of complexes of oligonucleotide-recombinant topoisomerases I analyzed on a 12% native polyacrylamide gel. The 22-mer oligonucleotide was incubated with TmTopo I, EcTopo I, LC, and RC proteins as described under "Experimental Procedures." To see the complexes and the cleavage products on the same gel, the reactions were loaded on a 12% native polyacrylamide gel. Lane C corresponds to the oligonucleotide control without protein. Incubation was made at 37 °C for 30 min. Prior to loading, half of the reaction was treated with proteinase K and SDS (right wells).

 
Formation of Covalent Complexes—0.4 pmol of the 3'-end-labeled oligonucleotide was incubated with 4 pmol of protein in buffer A for 30 min at 37 °C in a final volume of 10 µl. The reaction was stopped by addition of 2.5 µl of concentrated Laemmli buffer (312.5 mM Tris, pH 6.8, 50% glycerol, 0.1% bromphenol blue, 10% SDS, and 250 mM DTT). The samples were heated at 95 °C for 3 min, and 10 µl were loaded onto a 12% polyacrylamide gel. After electrophoresis, the gel was subjected to autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Rationale for the Design of Chimeras between T. maritima and E. coli Topoisomerases I—To evaluate the respective roles of the core and carboxyl-terminal regions in the mechanism of the T. maritima topoisomerase I compared with that of E. coli, chimeric proteins were constructed (Fig. 1). First, a truncated mutant composed of the first 540 amino acids of the T. maritima enzyme, TmTop65, was chosen from sequence comparisons with other bacterial topoisomerases I (10). It corresponds to the trans-esterification domain (core) of the protein and possesses all the conserved motifs present in the family. The core domain of E. coli topoisomerase I (the first 596 amino acids), called Top67, whose three-dimensional structure is known (8), was also reconstructed (see "Experimental Procedures") and cloned into the pET29b expression system as were the other constructions. The polypeptide produced under these conditions was renamed EcTop67 in order to avoid any confusion with TmTop65. The three chimeric proteins were then designed. In the first, named "long chimera," the entire carboxyl-terminal part of E. coli topoisomerase I (last 268 amino acids) containing the three zinc fingers was added to TmTop65. Because the two first zinc fingers in E. coli Topo I were described as essential for activity, a second chimera named "short chimera" was built, in which a sequence containing only the third zinc finger and the rest of the carboxyl-terminal domain of E. coli Topo I (157 amino acids) was fused to TmTop65. Finally, to look for a possible complementation of the inactive E. coli Top67 fragment by the carboxyl-terminal part of T. maritima topoisomerase I, a third chimeric protein, named "reverse chimera," was constructed in which the entire carboxyl-terminal sequence of T. maritima topoisomerase I (the last 93 residues) was fused to EcTop67.

Overexpression, Purification, and Zinc Content of the Recombinant Proteins—Eight proteins were overexpressed in E. coli and purified as described under "Experimental Procedures" as follows: the two full-length E. coli and T. maritima topoisomerases I, the Y288F inactive T. maritima mutant, in which the tyrosine involved in the trans-esterification reaction has been replaced by a phenylalanine (4), and the five constructs above described. Purified proteins were verified by gel electrophoresis under denaturing conditions. They exhibited the expected size and purity (Fig. 2). For the E. coli enzyme, we observed a doublet band, a result also obtained in other studies but did not appear to affect its properties (23).



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  		FIG. 2.
SDS-PAGE of the purified recombinant topoisomerases I. Approximately 3 µg of the different proteins were electrophoresed through a 12% SDS-polyacrylamide gel. Their nature is indicated above each well as defined under "Experimental Procedures" and Fig. 1. TmY288F represents the inactive mutant of T. maritima topoisomerase I, in which the tyrosine of the active site has been replaced by a phenylalanine. M are the molecular weight markers.

 
Previous studies stressed the importance of zinc fingers on the activity of E. coli topoisomerase I. We thus verified the ability of the various constructs to bind zinc atoms. The zinc content was titrated by using the sulfhydryl reagent PMPS. This compound is known to release zinc(II) from zinc-binding proteins by fixation of a mercury atom on cysteine residues (22). Released zinc is then fixed by the metallochrome indicator PAR (see "Experimental Procedures"). The results, shown in Fig. 3, indicate that in all cases the recombinant proteins produced in E. coli have incorporated the expected number of zinc atoms in their structure as follows: three zinc atoms are bound to the full-length E. coli enzyme and the LC, one zinc atom for full-length T. maritima enzyme, SC and RC, and no zinc atoms for TmTop65 and EcTop67.



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  		FIG. 3.
Zinc content titration of recombinant topoisomerases I. In each case, increasing amounts of PMPS were added to a 500-µl sample of 10 µM protein, as described under "Experimental Procedures." The control curve corresponds to zinc titration of buffer without enzyme.

 
T. maritima Topoisomerase I Lacking Its Carboxyl-terminal Part (TmTop65) Retains Low Relaxation Activity—The core domain of E. coli topoisomerase I (EcTop67) contains all the conserved motifs found in topoisomerases IA but lacks the ability to perform relaxation (1, 24). This prompted us to check the relaxation activity of the T. maritima topoisomerase deletion mutant TmTop65, equivalent to EcTop67. As shown in Fig. 4, the activity of TmTop65 is reduced about 250-fold compared with the full-length enzyme at the same temperature of 37 °C. This result emphasizes the importance of the carboxyl-terminal domain for enzyme activity, possibly by increasing its DNA binding efficiency (see binding experiments, Fig. 5).



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  		FIG. 4.
DNA relaxation activity and thermostability of the recombinant topoisomerases I. Proteins were preincubated (except in the left panels) at 50 °C for indicated times, prior to being diluted and tested in a standard relaxation assay at 37 °C as described under "Experimental Procedures." On each panel, serial dilutions were performed from 4 to 4 and corresponding to 1 pmol (lanes 1), 250 fmol (lanes 2), 60 fmol (lanes 3), 15 fmol (lanes 4), 4 fmol (lanes 5), 1 fmol (lanes 6), and 0.25 fmol (lanes 7) of protein. C represent the negatively supercoiled pTZ 18 DNA plasmid incubated without protein. FI, negatively supercoiled DNA; FII, open circular DNA.

 



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  		FIG. 5.
Oligonucleotide gel mobility shift assay of recombinant topoisomerases I. 0.2 pmol of the 5'-end-labeled 22-mer oligonucleotide was incubated with 4 pmol of full-length (TmTopo I, EcTopo I, and TmY288F), truncated (TmTop65 and EcTop67), and chimeric (LC, SC, and RC) topoisomerases as described under "Experimental Procedures." The reactions were processed and resolved through a 6% native polyacrylamide gel. No enzyme was added in the control reaction (lane C).

 
The observation that TmTop65 retains some relaxation activity was unexpected (Fig. 4 and Fig. 6, 0 panel) In the same experiment, EcTop67 is totally inactive, as described previously (1). This result suggests that the core domain TmTop65 of T. maritima is able to perform complete topoisomerization cycles, including strand passage and religation steps. Finally, because the optimal temperature for T. maritima topoisomerase is 75 °C, we also checked the activity of TmTop65 at this temperature and found that it is totally inactive at 75 °C (not shown).



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  		FIG. 6.
Determination of the cleavage site specificity of recombinant topoisomerases I. A, cleavage of the 5'-end-labeled 22-mer oligonucleotide by full-length (TmTopo I, EcTopo I, and TmY288F), truncated (TmTop65 and EcTop67), and chimeric proteins (LC, SC, and RC). The reactions were processed as described under "Experimental Procedures" and resolved on an 18% urea-polyacrylamide gel. No enzyme was added in the control reaction (lane C). M represents the oligonucleotide size markers. B, mapping of cleavage sites. Arrows indicate the cleavage positions obtained in A.

 
Thermal Stability and Relaxation Activity of the Chimeras— The results obtained with TmTop65 prompted us to verify the thermostability of the recombinant enzymes that we have created. All of them were inactivated at 75 °C (not shown), except the T. maritima full-length topoisomerase I (4). We then systematically investigated the relaxation activities of all the proteins at 37 °C and their thermostability after preincubation at 50 °C for various periods. The results are shown in Fig. 4. The first observation is that the mesophilic and the hyperthermophilic full-length topoisomerases exhibit comparable relaxation activities at 37 °C, although the distribution profile is different. At high concentrations of enzyme, the E. coli topoisomerase I fully relaxes DNA, whereas some topoisomers remain with the T. maritima enzyme. The second observation is that removal of the carboxyl-terminal domains, yielding EcTop67 and TmTop65, has dramatic effects on their activity and stability, stressing the importance of these domains for full activity.

The next question we addressed was the ability of a carboxyl-terminal domain to cross-activate the heterologous core enzyme. Starting with TmTop65, fusion of the complete carboxyl-terminal region of the E. coli Topo I restored an important activity at 37 °C. Indeed, the LC appears to be about 20-fold more active than TmTop 65. In addition, thermostability at 50 °C is partially restored; LC is still active after a 1-h incubation at this temperature, while TmTop65 is inactivated after 5 min. By contrast with LC, the SC poorly restores relaxation activity and remains unstable. These results indicate that the whole carboxyl-terminal domain of E. coli topoisomerase I is necessary to activate and stabilize a heterologous core domain.

We have also investigated the ability of the carboxyl-terminal part of T. maritima, fused to the totally inactive EcTop67, to restore activity. As shown in Fig. 4, a faint relaxation activity was reproducibly observed at 37 °C for this chimera. The activity disappears after 5 min at 50 °C, but the initial activity is probably too low to measure residual DNA relaxation. This experiment indicates that the core EcTop67 can be somewhat activated by the heterologous carboxyl-terminal domain of T. maritima topoisomerase I. The weak activity observed in this experiment parallels the faint increase in DNA binding (see below).

The Binding Efficiency of Both Topoisomerases Depends on the Presence of a Carboxyl-terminal Domain—Because the carboxyl-terminal portions of both E. coli and T. maritima topoisomerases appear essential to restore or stimulate the DNA relaxation activity, we further investigated the different steps of the topoisomerization cycle. The first step is the binding of the enzymes to DNA. For this analysis, a mobility shift experiment was performed, using the 22-mer single-stranded oligonucleotide previously described as a substrate exhibiting a unique site for T. maritima topoisomerase I (4) and labeled at its 5'-end (see "Experimental Procedures"). As shown in Fig. 5, both T. maritima and E. coli topoisomerases bind the oligonucleotide, producing a complex with low electrophoretic mobility in a native polyacrylamide gel. The mobility of the complex is the same as in the case of the Y288F mutant of T. maritima which is unable to cleave the oligonucleotide (Fig. 5, lane TmY288F). This point is important, because it shows that although the active recombinant enzymes presumably cleaved the oligonucleotide during incubation, the DNA fragments remain tightly associated in the complex. As is the case for DNA relaxation, removal of the carboxyl-terminal tails yielding Ec-Top67 and TmTop65 produces in both cases dramatic effects on oligonucleotide binding (Fig. 5). This result suggests that the main reason for the low activity or the complete lack of activity for TmTop65 and EcTop67, respectively, is the poor binding or the absence of binding to the DNA substrate.

We next evaluated the ability of a carboxyl-terminal domain to restore the binding of the heterologous core enzyme. This is evident for the long chimera where the addition of the carboxyl-terminal domain of E. coli topoisomerase to TmTop65 restores full binding capacity (Fig. 5, lane LC). Again, as for DNA relaxation, the short chimera exhibits poorly efficient binding, although a faint complex is reproducibly obtained (Fig. 5, lane SC). Finally, restoration of a low level of binding was also observed in the reverse chimera when the carboxyl-terminal part of T. maritima was fused to EcTop67 (Fig. 5, lane RC).

Together, these results indicate that the level of binding of recombinant proteins to single-stranded DNA parallels the DNA relaxation activity described above. Thus, binding to DNA, an event that constitutes the first step of the topoisomerization cycle, is an important determinant for activity and is stimulated by the carboxyl-terminal domain of the recombinant topoisomerases.

The Chimeric Protein (LC) between TmTop65 and E. coli Carboxyl-terminal Domain Predominantly Exhibits E. coli Topoisomerase I Cleavage Specificity—To analyze the ability of the different enzymes to cleave DNA and the specificity of cleavage, half of the incubation products observed in mobility shift experiments was loaded on a denaturing 18% urea-polyacrylamide gel. The results are shown in Fig. 6. All of the constructs, except the Y288F mutant, are able to cleave the oligonucleotide, although with different efficiencies and specificities. First, the full-length enzymes TmTopo I and EcTopo I both exhibit a unique cleavage point, with comparable cleavage intensity and preference for cytidine in position –4. However, their specificity appears to be different; TmTopo I cleaves the oligonucleotide after cytidine 17, whereas EcTopo I cleaves after guanosine 20 (Fig. 6B). Removal of the carboxyl-terminal parts dramatically reduces the cleavage level and changes the cleavage specificities (Fig. 6, lanes TmTop65 and EcTop67). TmTop65 cleaves after adenosine 13, and EcTop67 cleaves after cytidine 14 with low efficiency. This specificity shift suggests that the positioning of the core domains TmTop65 and EcTop67 alone on the oligonucleotide is different from that of the full-length topoisomerases with their carboxyl-terminal domains. Nevertheless, the rule of a cytidine in position –4 of the cleavage point (25–27) is followed in all cases.

The cleavage patterns obtained with the chimeras are surprising. For the LC chimera, the cleavage point (A12), specific for TmTop65, disappeared to the benefit of two other sites as follows: a major site equivalent to that found for the full-length E. coli topoisomerase I (G20), and a minor site equivalent to that found for the full-length T. maritima topoisomerase I (C17). This indicates that the carboxyl-terminal part of E. coli topoisomerase I contains determinants necessary to direct the specificity of DNA cleavage. Moreover, this carboxyl-terminal domain appears to allow two different positionings of the LC enzyme on the oligonucleotide. One is the same used by E. coli topoisomerase I, and the other, more puzzling, is the same as the positioning directed by the carboxyl-terminal part of T. maritima topoisomerase.

For the SC chimera, examination of the cleavage pattern shows that it retains the specificity of TmTop65. This again points to the importance of the two first zinc fingers present in LC for cleavage specificity.

The Chimeric Protein (RC) between EcTop67 and the T. maritima Carboxyl-terminal Domain Appears Unable to Hold the Fragments Produced by Oligonucleotide Cleavage—An unexpected result was the strong cleavage level obtained with the RC when the carboxyl-terminal part of T. maritima is added to EcTop67. Moreover, five cleavage sites, all different from those described above, were observed (Fig. 6A, lane RC), and none of them follow the cytidine –4 rule (Fig. 6B). This result contrasts with the very low level of binding to the same oligonucleotide (see Fig. 5) that would normally lead to poor cleavage. An explanation could be that the ability of DNA religation of this chimera is very low, in such a way that the 3'-end of the cleaved oligonucleotide is not retained by the protein. To verify this hypothesis, we analyzed the reactions used for the experiment of Fig. 5 on a more concentrated (12%) nondenaturing polyacrylamide gel to observe products migrating faster than the oligonucleotide. Samples were directly loaded or treated by SDS and proteinase K prior loading on the gel. The results are shown in Fig. 7. As already observed, TmTopo I, EcTopo I, and the chimera LC yielded slow migrating complexes with the 22-mer oligonucleotide. These complexes disappeared after treatment with SDS and proteinase K to the benefit of free DNA fragments migrating faster than the oligonucleotide (right wells of Fig. 7). These fragments correspond to cleavage products observed in Fig. 6A. However, the result is completely different in the case of the reverse chimera, for which broad bands were produced below the 22-mer substrate (Fig. 7, left wells). This pattern was unchanged upon treatment by SDS and proteinase K (Fig. 7, right wells). These results suggest that, contrary to the other topoisomerase constructs, RC is not able to hold the cleavage products noncovalently bound to the 3'-side of the breaks. This outcome could be the consequence of its multiple cleavage points on the oligonucleotide and addresses the question of covalent binding to the 5'-side. To gain further insight on the fate of the covalent complexes produced by the chimeric topoisomerases, the oligonucleotide substrate was labeled at its 3'-end, and the transfer of the label to the proteins was analyzed by SDS-PAGE (Fig. 8). As expected, the label appears associated with the full-length E. coli and T. maritima Topo I, as well as with LC. However, in the case of RC, no covalent adduct was detected. Additional experiments (see Supplemental Material) tended to favor the hypothesis of a hydrolytic activity of the RC preparation. In particular, we showed (Appendix 2) that the 5'-labeled fragments produced by RC are generated with much slower kinetics than are the cleavage products by TmTopo I and Ec Topo I. In addition, no fragment was produced by RC on a 3'-dideoxynucleotide-labeled substrate (Appendix 1), possibly because this oligonucleotide lacks a 3'-hydroxyl terminus.



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  		FIG. 8.
Formation of covalent complexes between the 3'-end-labeled 22-mer oligonucleotide and recombinant topoisomerases I. The labeled oligonucleotide was incubated with TmTopo I, LC, RC, EcTopo I, and TmY288F mutant, as described under "Experimental Procedures." Reaction products were separated on a 12% SDS-polyacrylamide gel and autoradiographed. Lane C corresponds to the oligonucleotide control incubated without protein. Molecular weight markers are indicated on the left.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The experiments described in this work allow a better understanding of the respective roles of the two domains distinguished in bacterial topoisomerases I as follows: the highly conserved core domains, also named trans-esterification domains, and the more divergent carboxyl-terminal regions. Different chimeras between E. coli and T. maritima domains were constructed and tested for their ability to bind, relax, and cleave DNA. The results are summarized in Table I.


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  		TABLE I
Summary of the results obtained with the different recombinant proteins

 
The first obvious result is the importance of the carboxyl-terminal domain of T. maritima topoisomerase I to promote high relaxation activity. Thus, this short domain of 93 amino acids might play a role similar to the large carboxyl-terminal domain of the E. coli enzyme. An unexpected observation was that the core enzyme TmTop65 of T. maritima is nevertheless able to perform relaxation of supercoiled DNA, although with low efficiency. Indeed, relaxation activity implies that the protein performs the following four successive steps in a topoisomerization cycle: binding to DNA, cleavage, strand passage, and religation. The level of relaxation activity mostly depends on the rate-limiting step. Here the low binding efficiency observed with TmTop65 parallels the low activity of cleavage and relaxation, suggesting that the limiting step is the first step, binding to DNA. Furthermore, addition of the carboxyl-terminal part of E. coli Topo I to TmTop65 increases both binding and relaxation activity by about 20-fold. Thus, we propose that the core TmTop65 domain alone is capable of performing a complete reaction of topoisomerization, including strand passage, and that the efficiency of the reaction is highly dependent on its binding to DNA substrate. The question remains whether this conclusion can be extrapolated to the core domains of other bacterial topoisomerases I. In the case of E. coli, DNA binding and relaxation activity become detectable after fusion of EcTop67 to the carboxyl-terminal domain of TmTopo I. It is noteworthy that this domain is poorly efficient, presumably because it incorrectly interacts with the EcTop67 domain. By comparison, for the full-length T. maritima topoisomerase I, the carboxyl-terminal domain likely interacts with its homologous core domain TmTop65, producing high DNA binding and relaxation activity. This role of the carboxyl-terminal part of T. maritima topoisomerase I differs from the generally admitted one of a carboxyl-terminal region playing a major role in DNA strand passage, the core domain being essentially in charge of the trans-esterifications (6, 20).

The information provided by the cleavage experiments support a major role of the carboxyl-terminal parts of the proteins in the positioning of the enzymes on DNA. Indeed, the cleavage obtained with the LC shows that the carboxyl-terminal part of E. coli topoisomerase I (268 amino acids) is able to confer the predominant cleavage specificity of the full-length E. coli enzyme (cleavage at guanosine 20) to the core domain TmTop65. The observation of a secondary cleavage point, corresponding to the specificity of the full-length T. maritima topoisomerase I (cleavage at cytidine 17), suggests two possibilities of positioning of the hybrid topoisomerase on the oligonucleotide. Another argument for an important role of the carboxyl-terminal domains in substrate recognition is that cleavage specificities of the core domains TmTop65 and EcTop67 are different from those of the full-length corresponding topoisomerases.

Previous results concerning the role of the carboxyl-terminal part in the reaction mechanism of E. coli topoisomerase I are not clear; consistent with our results, it has been shown that the enzyme, mutated in one of the three zinc fingers located in the carboxyl-terminal part of the protein, changed its cleavage site specificity (19). However, our conclusions differ from that of Ahumada et al. (20) who showed that E. coli Top67 had the same sequence and structure selectivity for DNA cleavage as the full-length enzyme. We cannot explain this discrepancy, although it could possibly be due to the nature of substrates used. Experiments with the hybrid topoisomerase SC, in which only the E. coli topoisomerase I region downstream of the second zinc finger is fused to TmTop65, suggest that the integrity of the carboxyl-terminal region is important for binding and substrate recognition. Indeed, the SC hybrid still exhibits poor binding, low cleavage, and low relaxation activity, similar to TmTop65. Moreover, the specificity of cleavage at adenosine 12 remains that of TmTop65. These results confirm the importance of the two first zinc fingers in the function of the E. coli carboxyl-terminal domain.

The results obtained with the RC chimera were puzzling. Indeed, whereas fusion of the carboxyl-terminal part of TmTopo I to EcTop67 weakly restored binding and relaxation activity, a high level of cleavage was observed, and five new sites were revealed that, contrary to the other constructions, lost the C-4 rule. In this case, it appears that the cleaved fragments are held neither on the 3'-side (usually noncovalently) nor on the 5'-side (usually covalently). Only a very low proportion of the enzyme is presumably engaged in a steady-state cleavable complex, accounting for the faint relaxation observed with RC. Two hypotheses can be proposed for the above observations. (i) The covalent adducts formed with RC are prone to hydrolysis, a property that is intrinsic to the chimera or the consequence of its numerous cleavage sites on the oligonucleotide that produce fragments of small size. (ii) the RC preparation contains a nuclease activity; the data included in the Supplemental Material support this hypothesis, showing an accumulation of DNA fragments over time. Whether this activity belongs to RC or to a contaminating exonuclease is not clear. Another important result was the ability of the heterologous E. coli carboxyl-terminal domains to confer some thermostability to TmTop65. This can be compared with the high stability of the full-length TmTopo I containing its homologous carboxyl-terminal domain. An additional role of the carboxyl-terminal parts in topoisomerases IA is perhaps to allow the protein to fold into a more stable structure, and this stabilization is probably essential for a thermophilic topoisomerase. The two domains have to be intimately folded within the structure. This was confirmed in the case of TmTopo I by the protection to proteolytic digestion conferred by the presence of the carboxyl-terminal part of the enzyme.2

Taken together, the conclusions of the present work lead us to propose that the trans-esterification domain of topoisomerases IA is potentially able to perform complete topoisomerization cycles, with low efficiency and low substrate specificity. The role of the carboxyl-terminal domain can tentatively be compared with that of an activator domain for RNA polymerase; it likely stabilizes the core domain, considerably increasing its binding efficiency to DNA and conferring more specific substrate recognition. Whether these findings can be extended to the entire topoisomerase IA family is still a possibility.


   FOOTNOTES
 
* This work was supported in part by the CNRS and the Université Paris-Sud XI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains Appendix 1 and Appendix 2. Back

{ddagger} Supported by a fellowship from the Ministère de la Recherche. Present address: Dept. of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, CA 94720-3204. Back

§ To whom correspondence should be addressed. Tel.: 33-1-69-15-46-19; Fax: 33-1-69-15-72-96; E-mail: bouthier{at}igmors.u-psud.fr.

1 The abbreviations used are: TmTopo, T. maritima topoisomerase I; PMPS, p-[hydroxymercuri]benzene sulfonate; LC, long chimera; RC, reverse chimera; SC, short chimera; PAR, 4-[2-pyridylazo]resorcinol; BSA, bovine serum albumin; DTT, dithiothreitol. Back

2 T. Viard, R. Cossard, M. Duguet, and C. Bouthier de La Tour, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. C. Serre for critical reading of the manuscript.



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 DISCUSSION
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