Cloning of cDNA Encoding a Novel Mouse DNA Topoisomerase III (Topo IIIβ) Possessing Negatively Supercoiled DNA Relaxing Activity, Whose Message Is Highly Expressed in the Testis*

We cloned cDNA encoding a novel mouse homologue of DNA topoisomerase III (mTOP3β). The nucleotide sequence contains an open reading frame of 863 amino acids, and the deduced molecular mass of the coded protein is 96.9 kDa. The overall sequence of mTOP3β has a 48 and 36% identity with mouse TOP3α at the nucleotide and amino acid level, respectively. DNA topoisomerase IIIβ was expressed using a baculovirus expression system and purified. The purified DNA topoisomerase IIIβ had activity to relax negatively supercoiled DNA. Relaxation of supercoiled DNA was partial at 37 °C and complete relaxation was observed at higher temperatures. mTOP3βmRNA was strongly expressed in the testis and relatively strongly in the brain. The levels of TOP3β mRNA in the testis increased slightly 14 days and considerably 17 days after birth, when the cells in the pachytene phase begin to appear and increase.

DNA topoisomerases are classified as type I or type II enzymes (1). Recently, DNA topoisomerase III belonging to the type IA subfamily, which includes Escherichia coli Topo I 1 and Topo III and yeast Topo III (2)(3)(4), have been found in higher eukaryotic cells (5). The gene encoding yeast Topo III (TOP3) was identified in 1989 as a suppressor of mitotic recombination between repetitive sequences (4). The yeast top3 mutant has a slow growth phenotype as well as a hyperrecombination phenotype. It is speculated that Topo III has a significant role in the unlinking of parental strands at the final stage of DNA replication and/or in the dissociation of structures that could lead to recombination (6).
The slow growth and hyperrecombination phenotypes of yeast top3 mutants were suppressed by mutations in SGS1 gene whose product was shown to interact functionally and physically with Topo III (7). The sgs1 mutants also show a heperrecombination phenotype (8). Thus it is conceivable that yeast Topo III acts in conjugation with Sgs1. Recently, human homologues of the SGS1 gene, BLM and WRN, have been cloned by positional cloning being the genes responsible for Bloom's syndrome (BS) and Werner's syndrome (WS), respectively (9,10). BS patients suffer cancer predisposition, immunodeficiency, and male infertility. In the BS cells, the interchanges between homologous chromosomes are increased, and an abnormally large number of sister chromatid exchanges are present (11). WS is known as a disease that causes premature aging, and cells derived from patients show a reduced replicative life span and chromosome aberrations including deletion (10). In this context, mammalian Topo III is attracting attention.
A cDNA encoding human Topo III was cloned in 1996 (5). The gene disruption study of mouse TOP3 showed mammalian topo III was essential in early embryogenesis (12). Recently, a genomic sequence encoding a putative Topo III homologue was found within the human immunoglobulin gene locus (13). Thus, the Topo III encoded by the cloned cDNA and the putative Topo III in the genomic sequence were named by Li and Wang (12) as Topo III␣ and Topo III␤, respectively. However, nothing is known about Topo III␤.
In the course of cloning the mouse homologue to human TOP3␣, we cloned cDNA encoding a mouse homologue to human Topo III␤. In this study, we examined both whether Topo III␤ has DNA topoisomerase activity or not using a purified protein and also the expression of TOP3␤ mRNA in various mouse tissues and in the testis during postnatal development.

EXPERIMENTAL PROCEDURES
cDNA Cloning of TOP3␤-A human TOP3␣ cDNA was obtained by PCR using a human fetal brain cDNA library and primers, 5Ј-ATGAT-CTTTCCTGTCGCCCG-3Ј and 5Ј-TGTTCTGAGGACAAAAGGGAC-3Ј. The PCR product was used to make a digoxigenin-labeled probe by random priming (DIG DNA Labeling Kit, Boehringer Mannheim). The probe thus obtained was used to screen a mouse brain gt10 cDNA library (oligo(dT)-primed). Two positive clones were obtained from approximately 1 ϫ 10 6 clones, and Southern blot and sequencing analyses indicated that both of them encoded a protein that had homology with human Topo III␣ lacking the 5Ј-terminal region but not Topo III␣ itself. To obtain the 5Ј-terminal region, PCR on a T-cell line cDNA library was performed using primers, 5Ј-CTGTGAAGCCTGGAGAAA-TAACTG-3Ј and 5Ј-ATTGAGATGGTGCACGATGC-3Ј (the sequence derived from the vector), and gave a cDNA fragment (nucleotide numbers, 688 -1477). Additional PCR from the mouse spermatocyte cDNA library was performed using primers, 5Ј-ACCCAGTAGGTCTC-TGGCTTGAAG-3Ј and 5Ј-TTGACACCAGACCAACTGGTAATG-3Ј (the sequence derived from the vector), and a cDNA fragment (1-842) was obtained. These cDNA fragments were connected by PCR and subcloned in the pGEM-T Easy vector to generate mTOP3␤/pGEM. Finally, the * This work was supported by grants-in-aid for Scientific Research, for Scientific Research on Priority Areas, and for JSPS Fellows from The Ministry of Education, Science, Sports, and Culture of Japan and the Mitsubishi Foundation. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB013603.
Generation of a Recombinant Baculovirus Harboring mTOP3␤ cDNA-mTOP3␤ gene was amplified by PCR from mTOP3␤/pGEM-T using primers, 5Ј-GAGAATTCATGAAGACCGTGCTCATGGTAG-3Ј and M13 reverse primer and subcloned into a pFASTBAC-HTa transfer vector using the EcoRI site. A recombinant baculovirus DNA containing mTOP3␤ cDNA was constructed by a BAC-TO-BAC baculovirus expression system (Life Technologies, Inc.) in accordance with the instruction manual.
Assay for DNA Topoisomerase Activity-DNA topoisomerase activity was assayed by measuring the negatively supercoiled DNA relaxing activity. The standard reaction mixture (15 l) for the DNA relaxation assay consisted of 40 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 , 5 mM dithiothreitol, 0.1 mg/ml BSA, and 0.2 g of supercoiled pBR322 DNA. For the assay of the activity of Topo I, the reaction mixture consisted of 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 0.1 mg/ml BSA, 0.2 g of DNA, and 10 units of Topo I from HeLa cells. The incubation proceeded at 37°C unless otherwise indicated, and the reaction was stopped with 2 l of a stop solution (1% SDS, 50% glycerol, 0.05% bromphenol blue). The sample was then loaded onto a 0.8% agarose gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA). After electrophoresis, the gel was stained with ethidium bromide and photographed under UV illumination.
Positively supercoiled DNA was generated by using archaebacterial reverse gyrase in the reaction mixture containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , 1 mM dithiothreitol, 1 mM ATP, 0.2 M NaCl, 0.1 mg/ml BSA, 20 mg/ml negatively supercoiled pBR322 DNA, and 1.5 g/ml reverse gyrase at 55°C for 30 min. The DNA thus treated was purified and used for the following assay. Assays were carried out with Topo III (150 ng) or Topo I (10 units) at 37°C for 15 min. Twodimensional electrophoresis was carried out as described previously (14).

RESULTS AND DISCUSSION
cDNA Cloning of mTOP3␤-In the course of cloning the mouse homologue to human TOP3␣, we isolated cDNA fragments encoding a protein that had homology to human TOP3␣ but was distinct from mTOP3␣. Thus, we cloned cDNA covering the full-length of the putative TOP3. The translation initiation site (AAGA ATG AAG) has a favorable context for efficient translation initiation, since there is an A residue at the Ϫ3 position and an A residue at the ϩ4 position (16). The nucleotide sequence contains an open reading frame of 863 amino acids, and the deduced molecular mass of the coded protein is 96.9 kDa. The deduced amino acid sequence showed homology to human and mouse Topo III␣ and Saccharomyces cerevisiae Topo III. Recently, a human genomic sequence containing a putative ORF encoding a protein possessing a homology with Topo III␣ has been reported and this gene was named TOP3␤ (12,13). Thus the deduced amino acid sequences were compared between the novel mouse TOP3 and the human TOP3␤. The result of alignment of amino acids using the BLAST program indicated that the mouse novel TOP3 has an 86% amino acid identity with the human TOP3␤ (Fig. 1). Thus we named the novel mouse TOP3 as mTOP3␤. The overall sequence of mTOP3␤ has a 48 and 36% identity with mTOP3␣ at the nucleotide and amino acid levels, respectively. The putative active site region of type IA topoisomerase is conserved in mTopo III␤ like other eukaryotic TOP3 gene products.
Purification of Recombinant mTopo III␤ and Detection of DNA Topoisomerase Activity-To examine whether the mTOP3␤ gene product has DNA topoisomerase activity, mTopo  4. Northern blotting of mTOP3␤ and mTOP3␣ mRNA using total RNA from various mouse tissues and from testes of various ages. RNA transferred to a membrane was hybridized with the mTOP3␣ probe, then the membrane was washed to strip the probe and rehybridized with the mTOP3␤ probe. Finally the blot was hybridized with the control probe of glycelaldehyde-3-phosphate dehydrogenase (G3PDH) after washing off the mTOP3␤ probe. A, Northern blot analysis of total RNA extracted from various mouse tissues. B, Northern blot analysis of total RNA extracted from testes of mice of various ages.
III␤ was expressed in insect cells using a recombinant baculovirus DNA harboring mTOP3␤ cDNA with a hexahistidine tag and was purified with an IMAC column. mTopo III␤ was eluted from the column at 100 mM imidazole by stepwise elution. An analysis by SDS-PAGE indicated that the purified fraction contained a single band of about 100 kDa (Fig. 2).
We assayed the DNA topoisomerase activity of mTopo III␤ using the purified fraction. A partial relaxation of negatively supercoiled DNA by mTopo III␤ was observed at 37°C. Fig. 3A shows the time course of the reaction. A partial relaxation of supercoiled DNA was observed after incubating for only 1 min. Although the amount of partially relaxed DNA was increased during incubation in a time-dependent manner, the change in the linking number of partially relaxed DNA was very slow, and the amount of completely relaxed DNA was very small even after incubation for 15 min (Fig. 3A, left panel). These results indicate that the relaxation of DNA becomes harder as the number of supercoiling of DNA decreases, namely, the chance to expose a single-stranded portion decreases.
DNA relaxing activity of mTopo III␤ was also confirmed by performing electrophoresis in a chloroquine-containing gel. As shown in the right panel of Fig. 3A, the amount of fast migrating DNA increased in a time-dependent manner.
We next assayed the enzyme activity at various temperatures from 37 to 62°C (Fig. 3B). The change in linking number was observed at all the temperatures tested up to 62°C. The number of DNA molecules that changed their linking number decreased upon rising incubation temperatures. However, complete relaxation of DNA became prominent at temperatures higher than 47°C. These results seem to indicate that mTopo III␤ becomes more unstable at higher temperatures, but once bound to DNA, it is stabilized and can continue relaxation of DNA, because the chance to expose a single-stranded portion increases at higher temperatures. Fig. 3C shows the magnesium requirement for the topoisomerase activity. In the presence of 5 and 10 mM Mg 2ϩ , more DNA molecules were relaxed than in the presence of 1 mM Mg 2ϩ or in its absence, but the levels of relaxation were lower under the former conditions, probably due to the difficulty in exposing a single-stranded portion. In the absence of Mg 2ϩ and in the presence of 1 mM EDTA, complete relaxation was observed in a small portion of supercoiled DNA. The reason why relaxation occurs in the absence of Mg 2ϩ is not clear at present. However, it is conceivable that a small portion of Topo III␤ still contains divalent cations under the above conditions and performs relaxation under the conditions relatively easy to expose a single-strand portion.
To confirm the requirement of a single-strand portion for mTopo III␤ to exhibit activity, positively supercoiled DNA was used as the substrate. mTopo III␤ was not able to relax positively supercoiled DNA which was relaxed by HeLa Topo I (Fig.  1D).
These results suggest that the preferable substrate for mTopo III␤, like yeast Topo III, might be a DNA containing a partially unwound region, which appears by the action of DNA helicases. In this respect, the report that yeast Topo III inter-acts with Sgs1 genetically and physically is interesting, because Sgs1 has DNA helicase activity (17).
Expression of mTOP3␤ mRNA-The expression of mTOP3␤ mRNA in various mouse tissues was examined by Northern blotting (Fig. 4A). An approximately 3-kilobase transcript was strongly expressed in the testis, relatively strongly in the brain, and weakly in other tissues including the heart, lung, thymus, and kidney. The mTOP3␣ mRNA was also expressed strongly in the testis, but in contrast to TOP3␤, very weakly in other tissues as reported previously (15).
We next examined the expression of TOP3␤ mRNA in the testis using RNA extracted from the testes of mice of various ages because spermatogenesis occurs fairly synchronously in the testis after birth. It was found that the TOP3␤ mRNA, similar to the TOP3␣ mRNA, increased slightly 14 days and considerably 17 days after birth, when the cells in the pachytene phase begin to appear and increase (Fig. 4B). These results appear to indicate that Topo III␤ is involved in some process of meiotic recombination probably with Topo III␣, because meiotic recombination is known to occur in pachytene cells. Interestingly, this expression pattern is similar to that of mBLM (18). Thus it appears likely that these proteins function in the same developmental stages in the testis.
Recently, it has been reported that Topo III␣ is essential in early embryogenesis in mice. It would be interesting to know whether Topo III␤ is essential for the viability of cells like as Topo III␣ or not. We are now trying to make TOP3␤-disrupted cells using a chicken B cell line, DT40 (19).