Mammalian DNA topoisomerase IIIa is essential in early embryogenesis (targeted gene disruptionyDtop3a miceygenome instability)

Targeted disruption of the mouse TOP3a gene encoding DNA topoisomerase IIIa was carried out to study the physiological functions of the mammalian type IA DNA topoisomerase. Whereas heterozygous top3a1/2 mutant mice were found to resemble phenotypically their TOP3a1/1 litermates, no viable top3a2/2 homozygotes were found among over 100 progeny of top3a1/2 intercrosses. Examination of embryos dissected from decidual swellings and in vitro culturing of blastocysts from top3a1/2 intercrosses showed that implantation of top3a2/2 embryos and the induction of decidualization could occur, but viability of these embryos was severely compromised at an early stage of development. The requirement of mouse DNA topoisomerase IIIa during early embryogenesis is discussed in terms of its plausible role in chromosome replication and its interaction with the RecQy SGS1 family of DNA helicases, whose members include the Bloom’s syndrome and the Werner’s syndrome gene products. Several recent studies of Bloom’s syndrome, Werner’s syndrome, and ataxia talangiectasia, three congenital diseases that exhibit elevated mitotic recombination rate and high incidence of cancer (1–3), have heightened interests on the physiological roles of mammalian DNA topoisomerase III (for reviews on DNA topoisomerases, see ref. 4 and references therein). The determinants of the first two, the BLM and WRN genes, encode proteins that are homologous to the budding yeast SGS1 gene product (1, 2), a DNA helicase that interacts with DNA topoisomerase III physically and functionally (5, 6). For human ataxia talangiectasia cells, which appear to lack a protein involved in cell-cycle regulation (7), overexpression of a truncated but not an intact DNA topoisomerase III was found to suppress their hyperrecombination phenotype (3). This finding was interpreted in terms of a recombinogenic DNA topoisomerase III in ataxia talangiectasia cells; thus overexpression of a truncated and inactive enzyme could exert a dominant negative effect (3). Mammalian DNA topoisomerase III (8) belongs to the type IA DNA topoisomerase subfamily whose members also include bacterial DNA topoisomerases I and III, yeast DNA topoisomerase III, and the enzyme ‘‘reverse gyrase’’ found in hyperthermophiles (4). Information on the physiological roles of the type IA DNA topoisomerases came mostly from studies of the bacterial and yeast enzymes. The intracellular level of Escherichia coli DNA topoisomerase I is intricately regulated by transcription from multiple promoters (9, 10), and inactivation of the enzyme is lethal in the absence of a compensatory mutation (11, 12). Because several of the compensatory mutations were found to map in genes encoding the subunits of DNA gyrase, it was thought that in vivo the removal of negative supercoils by E. coli DNA topoisomerase I would counter the negative supercoiling action of gyrase to maintain the proper degree of DNA supercoiling (11–13). This interpretation was modified through the proposal of the twin-supercoiled-domain model of transcription, which postulates that both positive and negative supercoils may be generated by transcription and other processes involving the tracking of macromolecular assemblies along DNA (14). According to this model, DNA gyrase, which effectively removes positive supercoils, and DNA topoisomerase I, which effectively removes negative supercoils, can be viewed as a cooperative pair that act jointly to solve the problem of excessive supercoiling of intracellular DNA (14). In the absence of E. coli DNA topoisomerase I, excessive negative supercoiling of intracellular DNA may lead to aberrant processes such as R-loop formation between nascent RNA and the DNA template (15, 16), and the cells may die as a consequence. E. coli DNA topoisomerase III, on the other hand, is dispensable; mutants lacking the enzyme exhibit a significantly higher recombination rate between repetitive sequences, however (17). In the budding yeast Saccharomyces cereviciae, there is only one type IA enzyme, DNA topoisomerase III. Yeast cells lacking the enzyme are viable, but their growth rate is reduced by nearly 2-fold and recombination between repetitive sequences is elevated substantially (18). In addition, yeast top32/2 diploid cells are unable to sporulate (18). Because of the low cellular level of DNA topoisomerase III in yeast and the presence of DNA topoisomerases I and II that are efficient in the removal of DNA supercoils, it is difficult to attribute the phenotypes of yeast top3 mutants to a loss of the supercoilremoval activity of DNA topoisomerase III (19–21). An alternative interpretation is that the yeast enzyme might have a significant role in the unlinking of parental strands at the final stage of chromosome replication andyor in the dissociation of structures in mitotic cells that could lead to recombination (5, 19–21). Because both the slow growth and hyperrecombination phenotype of yeast top3 mutants are suppressed by mutations in the SGS1 helicase gene, it is plausible that the DNA topoisomerase may act in conjunction with the SGS1 helicase in these processes (5). Whereas there is no direct evidence that yeast DNA topoisomerase III is a potent decatenase, it has been shown that purified E. coli DNA topoisomerase III, which resembles the yeast enzyme in several respects (21), is very efficient in unlinking intertwined parental strands in a plasmid DNA replication system (22). Human DNA topoisomerase III was identified in 1996 and its structural gene mapped to chromosome 17p11.2–12 (8). Recent nucleotide sequencing results have suggested, however, that there is a variant of the enzyme encoded by a gene within the Ig lambda locus at chromosome 22q11–12 (23). This putative variant enzyme is tentatively designated DNA topoisomerase IIIb and the activity reported earlier (8) as DNA topoisomerase IIIa. The likely presence of more than one type 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. © 1998 by The National Academy of Sciences 0027-8424y98y951010-4$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviation: dpc, days postcoitum. *To whom reprint requests should be addressed.