We cloned cDNA encoding a novel mouse homologue of DNA topoisomerase III (mTOP3␤). 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 mTOP3␤ has a 48 and 36% identity with mouse TOP3␣ at the nucleotide and amino acid level, respectively. DNA topoisomerase III␤ was expressed using a baculovirus expression system and purified. The purified DNA topoisomerase III␤ had activity to relax negatively supercoiled DNA. Relaxation of supercoiled DNA was partial at 37°C and complete relaxation was observed at higher temperatures. mTOP3␤ mRNA was strongly expressed in the testis and relatively strongly in the brain. The levels of TOP3␤ 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 (2-4), 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....

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A mutant allele of SGS1 of Saccharomyces cerevisiae was identified as a suppressor of the slow-growth phenotype of top3 mutants. We previously reported the involvement of Top3 via the interaction with the N-terminal region of Sgs1 in the complementation of methylmethanesulfonate (MMS) sensitivity and the suppression of hyper recombination of a sgs1 mutant. In this study, we found that several amino acids residues in the N-terminal region of Sgs1 between residues 4 and 33 were responsible for binding to Top3 and essential for complementing the sensitivity to MMS of sgs1 cells. Two-hybrid assays suggested that the region of Top3 responsible for the binding to Sgs1 was bipartite, with portion in the N-and C-terminal domains. Although disruption of the SGS1 gene suppressed the semilethality of the top3 mutant of strain MR, the sgs1-top3 double mutant grew more slowly and was more sensitive to MMS than the sgs1 single mutant, indicating that Top3 plays some role independently of Sgs1. The DNA topoisomerase activity of Top3 was required for the Top3 function to repair DNA damages induced by MMS, as shown by the fact that the TOP3 gene carrying a mutation (Phe for Tyr) at the amino acid residue essential for its activity (residue 356) failed to restore the MMS sensitivity of sgs1-top3 to the level of that of the sgs1 single mutant. Epistatic analysis using the sgs1-top3 double mutant, rad52 mutant and sgs1-top3-rad52 triple mutant indicated that TOP3 belongs to the RAD52 recombinational repair pathway.

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