Introduction DNA topoisomerases play important roles in DNA metabolism through their ability to catalyse the inter-conversion of topological isomers of DNA (Champoux, 2001; Wang, 1996; Watt and Hickson, 1994; Wigley, 1995). The fission yeast Schizosaccharomyces pombe expresses three topoisomerases, designated topoisomerases I, II and III. While the functions of topoisomerases I and II are quite well established, the role of topoisomerase III is not fully understood, in part because this class of enzyme possesses only weak DNA relaxation activity and is thought unlikely to participate in the maintenance of DNA supercoiling homeostasis (Goulaouic et al., 1999; Kim and Wang, 1992). The best-characterised top3 gene is from the budding yeast Saccharomyces cerevisiae. In this organism, deletion of Top3 results in hyper-recombination between repetitive DNA elements, slow growth due to a propensity to arrest at the G2-M DNA damage checkpoint, and defects in sporulation and S-phase responses to DNA damage (Chakraverty et al., 2001; Gangloff et al., 1999; Wallis et al., 1989). In contrast, deletion of top3 in S. pombe results in defective nuclear division and lethality (Goodwin et al., 1999; Maftahi et al., 1999). Two human topoisomerase III homologues, TOPOIIIα and TOPOIIIβ, have been identified (Hanai et al., 1996; Ng et al., 1999) and murine top3α has been shown to be essential for embryonic development (Li and Wang, 1998). Mice lacking top3β develop to maturity but show a reduced mean lifespan (Kwan and Wang, 2001). Accumulating evidence suggests that RecQ helicases act in concert with topoisomerase III. Interestingly, mutation of SGS1 or rqh1 + , which encode the sole RecQ homologues found in budding and fission yeast, respectively, can suppress the deleterious effects of loss of top3 function (Gangloff et al., 1994; Goodwin et al., 1999; Maftahi et al., 1999). There are five human RecQ-like helicase proteins: BLM, WRN, RECQL, RECQ4 and RECQ5. WRN is mutated in the premature ageing disorder Werner’s syndrome and RECQ4 is defective in Rothmund-Thomson syndrome (Kitao et al., 1999; Yu et al., 1996). Mutations in BLM cause Bloom syndrome (Ellis et al., 1995), the hallmark of which at the cellular level is an unusually high frequency of sister chromatid exchanges (Chaganti et al., 1974). RECQ5 physically interacts with TOP3α and TOP3β (Shimamoto et al., 2000) and the BLM protein binds to TOP3α (Wu et al., 2000), while overexpressed Caenorhabditis elegans TOP3α interacts physically with the RecQ homologue Him6 in vitro (Kim et al., 2002). Rqh1, the single S. pombe homologue, exists with Top3 in a high- molecular-weight complex (Laursen et al., 2003). The S. cerevisiae Sgs1 and Top3 proteins also interact physically, raising the possibility that Sgs1 may recruit Top3 to its site of action (Bennett and Wang, 2001). Several observations suggest that the function of the Top3- RecQ complex is required during S phase. S. cerevisiae sgs1 and top3 mutants are sensitive to hydroxyurea (HU), which blocks DNA replication by depletion of dNTP pools (Frei and Gasser, 2000; Mullen et al., 2000). In S. pombe, treatment of a temperature-sensitive top3 mutant with HU leads to increased 4769 In Schizosaccharomyces pombe, topoisomerase III is encoded by a single gene, top3 + , which is essential for cell viability and proper chromosome segregation. Deletion of rqh1 + , which encodes the sole RecQ family helicase in S. pombe, suppresses the lethality caused by loss of top3. Here, we provide evidence suggesting that the lethality in top3 mutants is due to accumulation of aberrant DNA structures that arise during S phase, as judged by pulsed-field gel electrophoresis. Using a top3 shut-off strain, we show here that depletion of Top3 activates the DNA damage checkpoint associated with phosphorylation of the checkpoint kinase Chk1. Despite activation of this checkpoint, top3 cells exit the arrest but fail to undergo faithful chromosome segregation. However, these mitotic defects are secondary to chromosomal abnormalities that lead to the lethality, because advance into mitosis did not adversely affect cell survival. Furthermore, top3 function is required for maintenance of nucleolar structure, possibly due to its ability to prevent recombination at the rDNA loci. Our data are consistent with the notion that Top3 has a key function in homologous recombinational repair during S phase that is essential for ensuring subsequent fidelity of chromosome segregation. Key words: DNA Topoisomerase III, DNA damage checkpoint, Nucleolus Summary Requirement for Schizosaccharomyces pombe Top3 in the maintenance of chromosome integrity Thein Z. Win 1 , Adele Goodwin 2 , Ian D. Hickson 2 , Chris J. Norbury 3 and Shao-Win Wang 1, * 1 Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK 2 Cancer Research UK Laboratories, University of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK 3 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK *Author for correspondence (e-mail: shaowin.wang@zoo.ox.ac.uk) Accepted 9 June 2004 Journal of Cell Science 117, 4769-4778 Published by The Company of Biologists 2004 doi:10.1242/jcs.01351 Research Article JCS ePress online publication date 31 August 2004