Human TOP 3 : A single - copy gene encoding DNA topoisomerase III ( sequence homology / chromosomal location / relaxation of supercoiled DNA / genome stability )

A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast Atopi cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17pll.2-12. In eukaryotes, enzymes belonging to each of the three subfamilies of DNA topoisomerases have been identified. Eukaryotic DNA topoisomerase I catalyzes the removal of positive and negative supercoils by transiently cleaving one DNA strand for the passage of another, and is thus classified as a type I topoisomerase (1). The enzyme appears to be the major cellular activity in the removal of supercoils generated by processes such as replication and transcription (2-4). Topl null mutants of the yeasts Schizosaccharomycespombe and Saccharomyces cerevisiae are viable (2, 3), and various experiments suggest that the dispensability of the enzyme is due to substitution of its cellular function by DNA topoisomerase II (5-7). In Drosophila, however, DNA topoisomerase I is essential, probably because of the rapid rate of DNA replication during early embryogenesis (8). Eukaryotic DNA topoisomerase II is a type II topoisomerase, which catalyzes the transient breakage of a doublestranded DNA and the transport of another DNA double helix through the break before its resealing (9-11). This enzyme catalyzes both the removal of positive and negative supercoils and the unlinking of intertwined pairs of DNA double helices. The latter activity cannot be substituted by type I topoisomerases, and thus a type II DNA topoisomerase is essential for disentangling DNA during condensation and mitotic and meiotic segregation of chromosomes (2, 3, 12-16). Eukaryotic DNA topoisomerase III'was discovered in 1989 (17). The gene encoding the yeast enzyme was originally identified by its suppression of mitotic recombination between repetitive sequences. Cloning and sequencing of this gene showed that it encodes a protein homologous to Escherichia coli DNA topoisomerase I, a type I enzyme that shares no significant sequence similarity with eukaryotic DNA topoisomerase I and was until then thought to exist only in prokaryotes. Based on the sequence data, the yeast gene was named TOP3, and its product, DNA topoisomerase III (17). Biochemical studies of purified yeast DNA topoisomerase III have confirmed the prediction that the protein, similar to E. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. coli DNA topoisomerase I, catalyzes the removal of negative but not positive supercoils (18). Both in vitro and in vivo studies suggest that relative to yeast DNA topoisomerases I and II, yeast DNA topoisomerase III has a much weaker activity in the removal of negative supercoils (18). It is therefore surprising that top3TOP1+ TOP2+ cells lacking the weak supercoilremoval activity ofDNA topoisomerase III but possessing two robust DNA topoisomerases I and II are nevertheless hyperrecombinogenic or hyper-rec and grow 50% more slowly relative to otherwise isogenic strains (17). Sporulation is also known to be completely blocked in top3 -/top3diploids (17). Recently, inactivation of a yeast gene SGS1 was found to suppress top3phenotypes (19). Sequence analysis indicates that SGS1 protein is homologousto E. coli RecQ protein, which possesses a DNA helicase activity and is involved in the RecF pathway of recombination (19). TOP3 and SGS1 proteins also appear to physically interact (19), which is reminiscent of the association between a bacterial DNA topoisomerase I-like domain and a helicase-like domain in the enzyme "reverse gyrase," an activity found in hyperthermophiles that positively supercoils DNA (20). Yeast TOP3 gene was also found recently (21) to be abutted head-to-head to a gene EST1, mutations in the latter lead to progressive shortening of telomeric repeats and cell senescense (22). The initiation codons of the divergent pair of genes are separated by 258 bp, and this close spacing suggests that the two might be coregulated (21). Mutations in TOP3 itself shorten telomeric repeats as well as destabilize subtelomeric elements (21). Whether these effects are related to EST1 function or they represent a manifestation of the general hyper-rec phenotype of top3 mutants is uncertain. Whereas DNA topoisomerases I and II are known to be ubiquitous and have been studied extensively, no other eukaryotic DNA topoisomerase III was reported since the 1989 finding of the yeast enzyme. Because of the known phenotypes of yeast DNA topoisomerase III and the general relation between genome stability and genetic disorders including cancer, we decided to examine whether multicellular organisms also possessed such an enzyme, and if so, whether the activity might be involved in suppressing mitotic recombination. In this communication, we report the identification of human DNA topoisomerase III.t MATERIALS AND METHODS Cloning and Characterization of Human TOP3 cDNA. The GenBank expressed sequence tag data base was first searched for reading frames that shared sequence similarities with yeast DNA topoisomerase III, using the Blast algorithm (23). Among the candidates identified, a 224-nt-long entry R45840 (24) had the potential of coding for a polypeptide with a heptameric stretch Gly-Ile-Gly-Thr-Asp-Ala-Thr identical to residues 544 to 550 of the yeast enzyme. The extent of similarity outside this heptameric identity was limited, but IThe sequence reported in this paper has been deposited in the GenBank data base (accession no. U43431).