Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase III␣ (Top3␣). However, the physiological relevance of the interaction between BLM and Top3␣ within the cell remains unclear. We show here that Top3␣ depletion causes accumulation of cells in G 2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3␣, these data indicate that BLM and Top3␣ execute the dissolution of sister chromatids.Eukaryotic TOP3 was first identified in Saccharomyces cerevisiae as a gene that is required to suppress recombination between repeated sequences (28). Deletion of TOP3 results in a slow-growth phenotype that is suppressed by the disruption of SGS1, the gene encoding the sole RecQ helicase in S. cerevisiae (5). Further analyses revealed that the function of Sgs1 is closely associated with that of DNA topoisomerase III (Top3) (2,13,19,27). The close relationship between RecQ helicases and Top3 seems to be maintained in higher eukaryotes. Higher eukaryotic cells have two Top3s, Top3␣ and Top3␤ (8,22,23). Knocking out the Top3␣ gene in mice results in embryonic lethality (17), while knocking out Top3␤ does not affect development but reduces the life span (15). Various Top3 and RecQ helicase molecules have been reported to interact physically, including Top3␣ and BLM (35), one of the RecQ family helicases in higher eukaryotic cells (3). BLM is a causative gene for Bloom's syndrome (3), which is an autosomal disorder characterized by predisposition to a wide variety of cancers (6). Biochemical analyses have suggested that BLM and Top3␣ together affect the in vitro resolution of a recombination intermediate containing a double Holliday junction (HJ) via a double-junction dissolution mechanism (34). However, the phenotypes of cells that lack Top3␣ have not been characterized precisely, since TOP3␣ knockout is lethal. Furthermore, the phenotypes of Top3␣-depleted cells before they die have not been examined. Moreover, the physiological relevance of the interaction between BLM and Top3␣ within the cell remains unclear. Therefore, elucidating higher eukaryotic Top3␣ function may enhance our understanding of the physiological roles of BLM.In this study, to assess the function of Top3␣ and its interactions with BLM, we constructed cells whose expression of Top3␣ can be switched off by doxycycline hydrochloride (Dox) treatment. To our knowledge, we present the first evidence to support the hypothesis that vertebrate Top3␣ together with the BLM helicase executes the dissolution of sister chromatids during DNA replication. MATERIALS AND METHODSPlasmid construction. Fragments of chicken TOP3␣ and TOP3␤ cDNAs ...

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system. INTRODUCTIONTo maintain genome integrity, chromosomes are precisely duplicated only once per cell division cycle. In eukaryotic cells, the replication licensing system ensures accurate DNA replication. The prereplication complex associates with origins of replication before S phase through the stepwise assembly of the origin recognition complex, Cdc6, Cdt1, and Mcm2-7, after which licensing takes place (Diffley, 2004;Blow and Dutta, 2006). Mcm2-7 is thought to act as the replicative helicase, and the loading of Mcm2-7 onto chromatin is considered to be the key initiating event of the licensing reaction (Pacek and Walter, 2004). Licensed origins are presumably activated in S phase by Cdc7-and cyclindependent kinase (CDK)-dependent processes, leading to the formation of replication forks and the recruitment of DNA polymerases.Repression of licensing after the onset of S phase is crucial for preventing rereplication (Fujita, 2006;Arias and Walter, 2007). In fission yeast, overexpression of Cdc18, an orthologue of Cdc6 in budding yeast and higher eukaryotes, induces rereplication (Nishitani et al., 2000;Yanow et al., 2001), suggesting that Cdc18/Cdc6 is a major target of mechanisms that repress licensing. In contrast, Cdt1 seems to be the crucial target in higher eukaryotes, because unregulated Cdt1 activity alone induces rereplication (Vaziri et al., 2003;Nishitani et al., 2004;Li and Blow, 2005;Arias and Walter, 2005;Maiorano et al., 2005). Geminin represses licensing by binding and inhibiting Cdt1 (Wohlschlegel et al., 2000;Tada et al., 2001), and it is inactivated or degraded on mitotic exit (McGarry and Kirschner, 1998;Li and Blow, 2004). The nuclear import of geminin during S phase reactivates it to restrict licensing (Hodgson et al., 2002;Yoshida et al., 2005). Crystallographic analysis revealed that geminin f...