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Human Topoisomerase IIIα Is a Single-stranded DNA Decatenase That Is Stimulated by BLM and RMI1

2010; Elsevier BV; Volume: 285; Issue: 28 Linguagem: Inglês

10.1074/jbc.m110.123216

ISSN

1083-351X

Autores

Jay Yang, Csanád Z. Bachrati, Jiongwen Ou, Ian D. Hickson, Grant W. Brown,

Tópico(s)

DNA and Nucleic Acid Chemistry

Resumo

Human topoisomerase IIIα is a type IA DNA topoisomerase that functions with BLM and RMI1 to resolve DNA replication and recombination intermediates. BLM, human topoisomerase IIIα, and RMI1 catalyze the dissolution of double Holliday junctions into noncrossover products via a strand-passage mechanism. We generated single-stranded catenanes that resemble the proposed dissolution intermediate recognized by human topoisomerase IIIα. We demonstrate that human topoisomerase IIIα is a single-stranded DNA decatenase that is specifically stimulated by the BLM-RMI1 pair. In addition, RMI1 interacts with human topoisomerase IIIα, and the interaction is required for the stimulatory effect of RMI1 on decatenase activity. Our data provide direct evidence that human topoisomerase IIIα functions as a decatenase with the assistance of BLM and RMI1 to facilitate the processing of homologous recombination intermediates without crossing over as a mechanism to preserve genome integrity. Human topoisomerase IIIα is a type IA DNA topoisomerase that functions with BLM and RMI1 to resolve DNA replication and recombination intermediates. BLM, human topoisomerase IIIα, and RMI1 catalyze the dissolution of double Holliday junctions into noncrossover products via a strand-passage mechanism. We generated single-stranded catenanes that resemble the proposed dissolution intermediate recognized by human topoisomerase IIIα. We demonstrate that human topoisomerase IIIα is a single-stranded DNA decatenase that is specifically stimulated by the BLM-RMI1 pair. In addition, RMI1 interacts with human topoisomerase IIIα, and the interaction is required for the stimulatory effect of RMI1 on decatenase activity. Our data provide direct evidence that human topoisomerase IIIα functions as a decatenase with the assistance of BLM and RMI1 to facilitate the processing of homologous recombination intermediates without crossing over as a mechanism to preserve genome integrity. IntroductionTopoisomerases are ubiquitous enzymes conserved from bacteria to humans. Their roles in modulating DNA topology in replication, transcription, and other cellular processes (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1870) Google Scholar) make them indispensable for cell viability. There are four subfamilies of topoisomerases as follows: IA, IB, IIA, and IIB. Type IA topoisomerases change DNA topological states in discrete steps of one via an enzyme-bridging mechanism (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1870) Google Scholar). The catalytic tyrosine residue initiates a transesterification reaction in a single-stranded region to generate a transient DNA break, allowing for the passage of the intact strand through the break. After religation of the broken strand by a reversal of the reaction, the enzyme is free to engage in another round of catalysis (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1870) Google Scholar). Members of the type IA topoisomerase family include Escherichia coli topoisomerase I (EcTop1) and III (EcTop3), yeast topoisomerase III (Top3), and two isoforms of topoisomerase III, α (Topo 2The abbreviations used are: TopotopoisomeraseDHJdouble Holliday junctionDTTdithiothreitolBSAbovine serum albuminhTopohuman topoisomeraseBSBloom syndromeSCEsister chromatid exchangeoligooligonucleotideGSTglutathione S-transferase. IIIα) and β (Topo IIIβ), in higher eukaryotes. These enzymes exhibit high sequence similarity in the N-terminal catalytic core domain, whereas the C-terminal tails are variable (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar). In addition to the ability to relax negatively supercoiled DNA, EcTop1 is capable of catalyzing knotting, unknotting, and interlinking of DNA substrates that contain exposed single-stranded regions (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar). Because single-stranded DNA gaps are a common feature found at DNA replication forks, replication termination sites, and replication and repair sites, it is believed that the main function of type IA topoisomerases is to unlink DNA catenanes.Type IA topoisomerases function in concert with RecQ helicases to control recombination events (3Mankouri H.W. Hickson I.D. Trends Biochem. Sci. 2007; 32: 538-546Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). RecQ helicases are a highly conserved family of DNA helicases that are required for the maintenance of genome integrity (4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). Human topoisomerase IIIα (hTopo IIIα) physically interacts with BLM, one of the five RecQ helicases in humans (5Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9644Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 6Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar). Biallelic mutations of BLM give rise to a clinically defined cancer predisposition disorder, Bloom syndrome (BS) (7German J. Dermatol. Clin. 1995; 13: 7-18Abstract Full Text PDF PubMed Google Scholar). BS cells display signs of genome instability, featuring an ∼10-fold elevation in the frequency of sister chromatid exchanges (SCE) (8Chaganti R.S. Schonberg S. German J. Proc. Natl. Acad. Sci. U.S.A. 1974; 71: 4508-4512Crossref PubMed Scopus (778) Google Scholar), events that arise from the processing of recombination intermediates (9Sonoda E. Sasaki M.S. Morrison C. Yamaguchi-Iwai Y. Takata M. Takeda S. Mol. Cell. Biol. 1999; 19: 5166-5169Crossref PubMed Scopus (365) Google Scholar). The hTopo IIIα interacting domain of BLM is required for suppression of SCE in BS cells (10Hu P. Beresten S.F. van Brabant A.J. Ye T.Z. Pandolfi P.P. Johnson F.B. Guarente L. Ellis N.A. Hum. Mol. Genet. 2001; 10: 1287-1298Crossref PubMed Google Scholar), suggesting that hTopo IIIα plays an anti-recombination role with BLM. Indeed, in vitro biochemical data show that BLM and hTopo IIIα catalyze the dissolution of double Holliday junctions (DHJs), a DNA structure that can arise as an intermediate during homologous recombination (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar). Dissolution occurs via a strand-passage mechanism that prevents genetic exchange between flanking sequences and is presumed to mimic the in vivo role of BLM-hTopo IIIα in suppressing SCE (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar). In the simplest case, the dissolution reaction is believed to have two components as follows: the helicase activity of BLM catalyzes branch migration of the Holliday junctions toward each other, resulting in collapse of the Holliday junctions, and generation of two duplex DNAs interlinked via catenated single strands. This structure, termed a hemicatenane, is then decatenated by hTopo IIIα to complete the dissolution of the DHJ (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar, 12Plank J.L. Wu J. Hsieh T.S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11118-11123Crossref PubMed Scopus (124) Google Scholar). Direct evidence that hTopo IIIα possesses the relevant decatenase activity, however, is currently lacking.RecQ helicases and type IA topoisomerases also cooperate to resolve converging replication forks. EcTop3 together with RecQ and the single-stranded DNA-binding protein SSB catalyzes the unlinking of late replication intermediates in vitro (13Suski C. Marians K.J. Mol. Cell. 2008; 30: 779-789Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). EcTop3 also catalyzes efficient decatenation of daughter DNA molecules during oriC and pBR322 replication in vitro (14Hiasa H. DiGate R.J. Marians K.J. J. Biol. Chem. 1994; 269: 2093-2099Abstract Full Text PDF PubMed Google Scholar). Although similar assays have not been performed within eukaryotes, several lines of evidence suggest that eukaryotic topoisomerase III functions in a similar role. First, Schizosaccharomyces pombe Top3 is required for normal chromosome segregation (15Goodwin A. Wang S.W. Toda T. Norbury C. Hickson I.D. Nucleic Acids Res. 1999; 27: 4050-4058Crossref PubMed Scopus (98) Google Scholar). Second, Topo IIIα depletion in chicken DT40 cells causes accumulation of metaphase cells with chromosome gaps and breaks (16Seki M. Nakagawa T. Seki T. Kato G. Tada S. Takahashi Y. Yoshimura A. Kobayashi T. Aoki A. Otsuki M. Habermann F.A. Tanabe H. Ishii Y. Enomoto T. Mol. Cell. Biol. 2006; 26: 6299-6307Crossref PubMed Scopus (42) Google Scholar). Finally, hTopo IIIα localizes to ultrafine anaphase DNA bridges in a BLM-dependent manner (17Chan K.L. North P.S. Hickson I.D. EMBO J. 2007; 26: 3397-3409Crossref PubMed Scopus (303) Google Scholar). In each of these cases, the failure of Topo III to decatenate and thereby resolve converging replication forks could lead to interlinked sister chromatids after replication and improper sister chromatid disjunction in mitosis.In eukaryotes, Topo III functions in concert with RMI (RecQ-mediated genomic instability) proteins (18Singh T.R. Ali A.M. Busygina V. Raynard S. Fan Q. Du C.H. Andreassen P.R. Sung P. Meetei A.R. Genes Dev. 2008; 22: 2856-2868Crossref PubMed Scopus (165) Google Scholar, 19Chang M. Bellaoui M. Zhang C. Desai R. Morozov P. Delgado-Cruzata L. Rothstein R. Freyer G.A. Boone C. Brown G.W. EMBO J. 2005; 24: 2024-2033Crossref PubMed Scopus (135) Google Scholar, 20Mullen J.R. Nallaseth F.S. Lan Y.Q. Slagle C.E. Brill S.J. Mol. Cell. Biol. 2005; 25: 4476-4487Crossref PubMed Scopus (131) Google Scholar, 21Raynard S. Bussen W. Sung P. J. Biol. Chem. 2006; 281: 13861-13864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar, 23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar, 24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar). In Saccharomyces cerevisiae, deletion of the gene encoding Rmi1 results in phenotypes similar to top3Δ, including sensitivity to DNA-damaging agents, and hyper-recombination (19Chang M. Bellaoui M. Zhang C. Desai R. Morozov P. Delgado-Cruzata L. Rothstein R. Freyer G.A. Boone C. Brown G.W. EMBO J. 2005; 24: 2024-2033Crossref PubMed Scopus (135) Google Scholar, 20Mullen J.R. Nallaseth F.S. Lan Y.Q. Slagle C.E. Brill S.J. Mol. Cell. Biol. 2005; 25: 4476-4487Crossref PubMed Scopus (131) Google Scholar), consistent with Rmi1 and Top3 functioning in the same pathway. In humans, RMI1 binds to hTopo IIIα via its conserved N-terminal domain (21Raynard S. Bussen W. Sung P. J. Biol. Chem. 2006; 281: 13861-13864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar), and this interaction appears to be important for hTopo IIIα stability in vivo (23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar). In vitro, RMI1 stimulates DHJ dissolution by hTopo IIIα and BLM (21Raynard S. Bussen W. Sung P. J. Biol. Chem. 2006; 281: 13861-13864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar). Although other type IA topoisomerases can function in DHJ dissolution in vitro, stimulation of dissolution by RMI1 specifically requires hTopo IIIα (22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar). RMI1 associates with a second RMI protein, RMI2, via the OB-fold domain at the C terminus to form a heterodimeric RMI complex (24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar). The RMI complex is required for the phosphorylation of BLM in mitotic cells and for the recruitment of BLM to nuclear foci in response to DNA damage (18Singh T.R. Ali A.M. Busygina V. Raynard S. Fan Q. Du C.H. Andreassen P.R. Sung P. Meetei A.R. Genes Dev. 2008; 22: 2856-2868Crossref PubMed Scopus (165) Google Scholar, 23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar, 24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar). Moreover, cells that are depleted of the RMI complex show an elevated level of SCE (23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar, 24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar), the hallmark of BS cells, indicating that the RMI complex is critical for BLM function. Thus, the complex of BLM-hTopo IIIα-RMI1-RMI2 likely represents the functional unit in higher eukaryotes and is termed the BLM core complex (25Liu Y. West S.C. Genes. Dev. 2008; 22: 2737-2742Crossref PubMed Scopus (27) Google Scholar).To define the biochemical role of hTopo IIIα in DHJ dissolution and in the resolution of converging replication forks, we have generated a catenane of single-stranded DNA circles to mimic the proposed target of hTopo IIIα in the final step of these reactions. Using an improved procedure for the expression and purification of hTopo IIIα, we demonstrate that hTopo IIIα acts as a single-stranded DNA decatenase in DHJ dissolution and that this decatenase activity is specifically stimulated by RMI1. Surprisingly, we also find that BLM stimulates decatenation, and that BLM and RMI1 together synergistically stimulate decatenation by hTopo IIIα. We propose that hTopo IIIα is the cellular decatenase that functions in dissolution of DHJs and resolution of converging replication forks and that the optimal decatenase activity depends on the formation of a complex with RMI1 as well as BLM helicase activity.DISCUSSIONUsing DNA substrates that resemble replication and recombination intermediates, numerous in vitro studies have made significant advances in elucidating the mechanistic details of RecQ-Topo III-mediated resolution of replication and recombination intermediates. Most notably, the helicase-topoisomerase partnership resolves converging replication forks (13Suski C. Marians K.J. Mol. Cell. 2008; 30: 779-789Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and resolves double Holliday junctions without crossing over (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar, 12Plank J.L. Wu J. Hsieh T.S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11118-11123Crossref PubMed Scopus (124) Google Scholar). In this study, we generated single-stranded catenanes that resemble the DNA structure that is likely recognized by hTopo IIIα in these reactions. We provide direct evidence that hTopo IIIα is a single-stranded DNA decatenase and that the decatenase activity is stimulated by members of the BLM core complex, BLM and RMI1. Modulation of decatenation and DHJ dissolution reactions requires physical interaction between RMI1 and hTopo IIIα. The specificity of single-stranded DNA decatenation and DHJ dissolution for type IA topoisomerases, as well as the specificity of the RMI1 stimulation of both reactions, indicates that the relevant activity of hTopo IIIα in the dissolution of DHJs is single-stranded DNA decatenation and not relaxation of superhelical stress.Improved Purification of hTopo IIIα and RMI1We developed improved purification methods for both hTopo IIIα and RMI1 by the use of S. cerevisiae as a host for expression. Several lines of evidence suggest that S. cerevisiae is a better tool than E. coli for the expression and purification of hTopo IIIα and RMI1. First, although RMI1 is insoluble in E. coli (22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar), the protein was soluble when overexpressed in S. cerevisiae. In addition, yeast-purified RMI1 was ∼5 times more active in stimulation of DHJ dissolution by BLM and hTopo IIIα (compare this study with Ref. 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar). hTopo IIIα purified from S. cerevisiae was also more active in both the decatenation (Fig. 2D and supplemental Fig. 6A) and relaxation assays (compare this study with Ref. 26Goulaouic H. Roulon T. Flamand O. Grondard L. Lavelle F. Riou J.F. Nucleic Acids Res. 1999; 27: 2443-2450Crossref PubMed Scopus (68) Google Scholar). Furthermore, in contrast to previous work with the yeast homologs Top3 and Rmi1, where stimulation of relaxation was only apparent when the proteins were co-expressed and co-purified (35Chen C.F. Brill S.J. J. Biol. Chem. 2007; 282: 28971-28979Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), we were able to reconstitute stimulation of hTopo IIIα from individually purified hTopo IIIα and RMI1 proteins in both DNA relaxation and decatenation assays. Our data indicate that expressing and purifying hTopo IIIα and RMI1 in S. cerevisiae yield functional proteins with optimal biochemical activities.Type IA Topoisomerases Are Single-stranded DNA DecatenasesWe have developed an elegant system to directly assay the single-stranded DNA decatenase activity of type IA topoisomerases. Previous work has indirectly assayed decatenation of type IA topoisomerases on complex substrates in the presence of RecQ helicases. Decatenase activity of type IA topoisomerases was demonstrated in an in vitro replication system in which EcTop3 converted plasmid replication intermediates into monomeric daughter molecules (14Hiasa H. DiGate R.J. Marians K.J. J. Biol. Chem. 1994; 269: 2093-2099Abstract Full Text PDF PubMed Google Scholar, 36Hiasa H. Marians K.J. J. Biol. Chem. 1994; 269: 32655-32659Abstract Full Text PDF PubMed Google Scholar). However, the complex nature of this system made it difficult to directly identify the DNA substrate on which the enzyme was acting. Recently, Suski and Marians (13Suski C. Marians K.J. Mol. Cell. 2008; 30: 779-789Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) used an improved system to trap replication intermediates right before replication forks converge, allowing for the study of enzyme activities at a more defined stage. Using this system, they demonstrated that RecQ and EcTop3 catalyze the resolution of converging replication forks via a strand-passage mechanism similar to that in DHJ dissolution, where RecQ-mediated DNA unwinding is followed by EcTop3-mediated decatenation to liberate plasmid DNAs with single-stranded gaps (13Suski C. Marians K.J. Mol. Cell. 2008; 30: 779-789Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Similarly, the second component of the DHJ dissolution activity reported by Wu and Hickson (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar) is proposed to be an hTopo IIIα-catalyzed decatenation of a hemicatenane. The simple single-stranded DNA catenane used here resembles the topoisomerase IA substrate at the latest stage in both the resolution and the dissolution reactions. Significantly, although both type IA and IB topoisomerases catalyze the relaxation of negatively supercoiled DNA by introducing a single-stranded nick (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar), the decatenation of single-stranded DNA reported here was specific to type IA topoisomerases. The specificity observed in the decatenation assay is consistent with that in the DHJ dissolution assay where type IA, but not type IB, topoisomerases are able to replace hTopo IIIα in catalyzing DHJ dissolution with BLM (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar). Both decatenation and DHJ dissolution require a type IA topoisomerase, and stimulation of both reactions by RMI1 requires the cognate topoisomerase hTopo IIIα. Stimulation of decatenation occurs at an hTopo IIIα:RMI1 stoichiometry near the expected 1:1 and is compromised in RMI1 mutants that bind hTopo IIIα poorly. Together, these data suggest that decatenation is the biologically relevant activity of hTopo IIIα in processing replication and recombination intermediates. Failure to decatenate these intermediates in vivo would generate interlinked sister chromatids and homologous chromosomes, leading to abnormal mitoses, chromosome breaks, and rearrangements. Consistent with this, hTopo IIIα localizes to ultra-fine DNA bridges during anaphase (17Chan K.L. North P.S. Hickson I.D. EMBO J. 2007; 26: 3397-3409Crossref PubMed Scopus (303) Google Scholar), and depletion of hTopo IIIα causes an increase in the number of DNA bridges (37Temime-Smaali N. Guittat L. Wenner T. Bayart E. Douarre C. Gomez D. Giraud-Panis M.J. Londono-Vallejo A. Gilson E. Amor-Guéret M. Riou J.F. EMBO J. 2008; 27: 1513-1524PubMed Google Scholar).BLM and RMI1 Cooperate with hTopo IIIα in DecatenationWe find that both BLM and RMI1 stimulate the decatenase activity of hTopo IIIα, although the underlying mechanisms of stimulation by the two proteins could be quite different. BLM stimulates the decatenase activity of both hTopo IIIα and EcTop1 in an ATP-dependent manner, suggesting that unwinding of the substrate by BLM activity contributes to the stimulation indirectly. However, the stimulation of EcTop1 decatenase activity is modest in comparison with the stimulation of hTopo IIIα (0.5- versus 2.5-fold), so it appears that a specific interaction of BLM with hTopo IIIα contributes to the stimulation. RMI1, on the other hand, is not able to promote EcTop1-mediated decatenation, and hTopo IIIα-binding mutants of RMI1 fail to stimulate hTopo IIIα-mediated decatenase activity. This strongly suggests that the stimulation occurs via specific interactions between hTopo IIIα and RMI1. We envisage two possible scenarios. In the first, RMI1 targets hTopo IIIα to the single-stranded catenane via a DNA binding activity of RMI1. Whether RMI1 has such a DNA binding activity, however, remains controversial. A DNA binding domain has been localized to the C terminus of human RMI1, but this domain is dispensable for the stimulation of DHJ dissolution (33Raynard S. Zhao W. Bussen W. Lu L. Ding Y.Y. Busygina V. Meetei A.R. Sung P. J. Biol. Chem. 2008; 283: 15701-15708Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and it seems unlikely that an activity critical for RMI1 function would be located outside of the conserved N-terminal region. Other studies of RMI1 failed to detect significant DNA binding activity (24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar). Budding yeast Rmi1, which lacks the C-terminal region, has weak single-stranded DNA binding activity and can bind Holliday junction structures (20Mullen J.R. Nallaseth F.S. Lan Y.Q. Slagle C.E. Brill S.J. Mol. Cell. Biol. 2005; 25: 4476-4487Crossref PubMed Scopus (131) Google Scholar, 35Chen C.F. Brill S.J. J. Biol. Chem. 2007; 282: 28971-28979Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It is possible that the N terminus of RMI1 contains a cryptic DNA binding activity that is activated upon binding to hTopo IIIα or that RMI1 recognizes catenated single-stranded DNA specifically. Alternatively, the interaction between hTopo IIIα and RMI1 could induce conformational changes in hTopo IIIα that enhance its decatenase activity. Consistent with this possibility, the Holliday junctions and DHJ binding activity of Topo III in yeast and human systems are stimulated by RMI1 (22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar, 35Chen C.F. Brill S.J. J. Biol. Chem. 2007; 282: 28971-28979Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar).Several lines of evidence have suggested that Topo III and RMI1 form a tight complex that might then have a more peripheral association with RecQ. For example, co-expressed Top3 and Rmi1 have increased solubility compared with the individual subunits (35Chen C.F. Brill S.J. J. Biol. Chem. 2007; 282: 28971-28979Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar); knockdown of RMI1 destabilizes hTopo IIIα, whereas knockdown of BLM does not (18Singh T.R. Ali A.M. Busygina V. Raynard S. Fan Q. Du C.H. Andreassen P.R. Sung P. Meetei A.R. Genes Dev. 2008; 22: 2856-2868Crossref PubMed Scopus (165) Google Scholar, 24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar); the Top3-Rmi1 complex binds to Sgs1 (19Chang M. Bellaoui M. Zhang C. Desai R. Morozov P. Delgado-Cruzata L. Rothstein R. Freyer G.A. Boone C. Brown G.W. EMBO J. 2005; 24: 2024-2033Crossref PubMed Scopus (135) Google Scholar, 35Chen C.F. Brill S.J. J. Biol. Chem. 2007; 282: 28971-28979Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar); deletion of rmi1 in yeast closely resembles the phenotype of top3 deletion rather than that of sgs1 deletion (19Chang M. Bellaoui M. Zhang C. Desai R. Morozov P. Delgado-Cruzata L. Rothstein R. Freyer G.A. Boone C. Brown G.W. EMBO J. 2005; 24: 2024-2033Crossref PubMed Scopus (135) Google Scholar, 20Mullen J.R. Nallaseth F.S. Lan Y.Q. Slagle C.E. Brill S.J. Mol. Cell. Biol. 2005; 25: 4476-4487Crossref PubMed Scopus (131) Google Scholar); stimulation of DHJ dissolution by RMI1 requires the cognate type IA topoisomerase, hTopo IIIα (22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar); and Topo IIIα and RMI1 perform meiotic functions that are independent of RecQ4a, a homolog of BLM helicase in Arabidopsis thaliana (38Hartung F. Suer S. Knoll A. Wurz-Wildersinn R. Puchta H. PLoS Genet. 2008; 4: e1000285Crossref PubMed Scopus (77) Google Scholar, 39Chelysheva L. Vezon D. Belcram K. Gendrot G. Grelon M. PLoS Genet. 2008; 4: e1000309Crossref PubMed Scopus (46) Google Scholar).In this context, and because other type IA topoisomerases can substitute for hTopo IIIα in DHJ dissolution, a stepwise model for the dissolution in which the helicase and the topoisomerase perform distinct roles has been proposed (40Cheok C.F. Bachrati C.Z. Chan K.L. Ralf C. Wu L. Hickson I.D. Biochem. Soc. Trans. 2005; 33: 1456-1459Crossref PubMed Scopus (102) Google Scholar). Although this is likely the case at the enzymatic level, because BLM lacks decatenase activity and hTopo IIIα lacks helicase activity, our data indicate that there is an important interplay between BLM, hTopo IIIα, and RMI1 even at the latest stage of the reaction where only decatenase activity is required. This interplay is apparent when the three proteins are incubated together in the decatenation assay. Although both BLM and RMI1 alone display modest stimulation on hTopo IIIα decatenase activity (by ∼2-fold), the BLM-RMI1 pair synergistically stimulates the activity by at least 20-fold. Moreover, this synergy depends on the physical interaction between RMI1 and Topo IIIα. Thus, the maximum level of decatenation requires all members of the BLM-Topo IIIα-RMI1 complex. The specificity for the entire BLM-Topo IIIα-RMI1 complex for optimal decatenation that we observe is also seen in Holliday junction unwinding by BLM, which requires hTopo IIIα-RMI1 for optimal activity (41Bussen W. Raynard S. Busygina V. Singh A.K. Sung P. J. Biol. Chem. 2007; 282: 31484-31492Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Because unwinding and decatenation both contribute to DHJ dissolution, it follows that maximum activity in DHJ dissolution is likewise only obtained when all three components are present (21Raynard S. Bussen W. Sung P. J. Biol. Chem. 2006; 281: 13861-13864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar). Furthermore, in a scenario where the two junctions are separated by multiple topologically constrained linkages, the activity of hTopo IIIα would likely be required at earlier stages to relieve superhelical stress generated by BLM-dependent branch migration. Therefore, although the steps in catalyzing DHJ dissolution appear to be conceptually distinct, the coordinated actions of all the members in the BLM core complex are likely required for optimal activity in suppressing SCEs in vivo. Finally, our results point to an added complexity in reactions catalyzed by RecQ-Topo III complexes in eukaryotes. Whereas work with late replication intermediates indicates functional cooperation between RecQ and Topo III in E. coli (13Suski C. Marians K.J. Mol. Cell. 2008; 30: 779-789Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), our data indicate that in eukaryotes the functional specificity and cooperation extend also to RMI1.The strand passage activity of BLM-hTopo IIIα-RMI1 is likely important for a number of cellular processes in which Holliday junctions are a feature (Fig. 8). These include the repair of DNA double-stranded breaks, the processing of stalled replication forks, the lengthening of telomeres via the alternative lengthening of telomeres pathway, and the resolution of converging replication forks, which are topologically similar to Holliday junctions. All of these processes share a common terminal step, decatenation. We propose that hTopo IIIα catalyzes the decatenation of these structures in vivo and that the decatenation is performed in cooperation with BLM and RMI1. Such a role is consistent with the recent association of two single-nucleotide polymorphisms in the hTopo IIIα gene with increased risk of cancer development (42Broberg K. Huynh E. Schläwicke Engström K. Björk J. Albin M. Ingvar C. Olsson H. Höglund M. BMC Cancer. 2009; 9: 140Crossref PubMed Scopus (35) Google Scholar). The details of decatenation catalyzed by hTopo IIIα presented here shed light on the role of hTopo IIIα and its cooperating partners BLM and RMI1 in the maintenance of genome integrity. IntroductionTopoisomerases are ubiquitous enzymes conserved from bacteria to humans. Their roles in modulating DNA topology in replication, transcription, and other cellular processes (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1870) Google Scholar) make them indispensable for cell viability. There are four subfamilies of topoisomerases as follows: IA, IB, IIA, and IIB. Type IA topoisomerases change DNA topological states in discrete steps of one via an enzyme-bridging mechanism (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1870) Google Scholar). The catalytic tyrosine residue initiates a transesterification reaction in a single-stranded region to generate a transient DNA break, allowing for the passage of the intact strand through the break. After religation of the broken strand by a reversal of the reaction, the enzyme is free to engage in another round of catalysis (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1870) Google Scholar). Members of the type IA topoisomerase family include Escherichia coli topoisomerase I (EcTop1) and III (EcTop3), yeast topoisomerase III (Top3), and two isoforms of topoisomerase III, α (Topo 2The abbreviations used are: TopotopoisomeraseDHJdouble Holliday junctionDTTdithiothreitolBSAbovine serum albuminhTopohuman topoisomeraseBSBloom syndromeSCEsister chromatid exchangeoligooligonucleotideGSTglutathione S-transferase. IIIα) and β (Topo IIIβ), in higher eukaryotes. These enzymes exhibit high sequence similarity in the N-terminal catalytic core domain, whereas the C-terminal tails are variable (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar). In addition to the ability to relax negatively supercoiled DNA, EcTop1 is capable of catalyzing knotting, unknotting, and interlinking of DNA substrates that contain exposed single-stranded regions (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2153) Google Scholar). Because single-stranded DNA gaps are a common feature found at DNA replication forks, replication termination sites, and replication and repair sites, it is believed that the main function of type IA topoisomerases is to unlink DNA catenanes.Type IA topoisomerases function in concert with RecQ helicases to control recombination events (3Mankouri H.W. Hickson I.D. Trends Biochem. Sci. 2007; 32: 538-546Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). RecQ helicases are a highly conserved family of DNA helicases that are required for the maintenance of genome integrity (4Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Crossref PubMed Scopus (576) Google Scholar). Human topoisomerase IIIα (hTopo IIIα) physically interacts with BLM, one of the five RecQ helicases in humans (5Wu L. Davies S.L. North P.S. Goulaouic H. Riou J.F. Turley H. Gatter K.C. Hickson I.D. J. Biol. Chem. 2000; 275: 9636-9644Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 6Johnson F.B. Lombard D.B. Neff N.F. Mastrangelo M.A. Dewolf W. Ellis N.A. Marciniak R.A. Yin Y. Jaenisch R. Guarente L. Cancer Res. 2000; 60: 1162-1167PubMed Google Scholar). Biallelic mutations of BLM give rise to a clinically defined cancer predisposition disorder, Bloom syndrome (BS) (7German J. Dermatol. Clin. 1995; 13: 7-18Abstract Full Text PDF PubMed Google Scholar). BS cells display signs of genome instability, featuring an ∼10-fold elevation in the frequency of sister chromatid exchanges (SCE) (8Chaganti R.S. Schonberg S. German J. Proc. Natl. Acad. Sci. U.S.A. 1974; 71: 4508-4512Crossref PubMed Scopus (778) Google Scholar), events that arise from the processing of recombination intermediates (9Sonoda E. Sasaki M.S. Morrison C. Yamaguchi-Iwai Y. Takata M. Takeda S. Mol. Cell. Biol. 1999; 19: 5166-5169Crossref PubMed Scopus (365) Google Scholar). The hTopo IIIα interacting domain of BLM is required for suppression of SCE in BS cells (10Hu P. Beresten S.F. van Brabant A.J. Ye T.Z. Pandolfi P.P. Johnson F.B. Guarente L. Ellis N.A. Hum. Mol. Genet. 2001; 10: 1287-1298Crossref PubMed Google Scholar), suggesting that hTopo IIIα plays an anti-recombination role with BLM. Indeed, in vitro biochemical data show that BLM and hTopo IIIα catalyze the dissolution of double Holliday junctions (DHJs), a DNA structure that can arise as an intermediate during homologous recombination (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar). Dissolution occurs via a strand-passage mechanism that prevents genetic exchange between flanking sequences and is presumed to mimic the in vivo role of BLM-hTopo IIIα in suppressing SCE (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar). In the simplest case, the dissolution reaction is believed to have two components as follows: the helicase activity of BLM catalyzes branch migration of the Holliday junctions toward each other, resulting in collapse of the Holliday junctions, and generation of two duplex DNAs interlinked via catenated single strands. This structure, termed a hemicatenane, is then decatenated by hTopo IIIα to complete the dissolution of the DHJ (11Wu L. Hickson I.D. Nature. 2003; 426: 870-874Crossref PubMed Scopus (868) Google Scholar, 12Plank J.L. Wu J. Hsieh T.S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11118-11123Crossref PubMed Scopus (124) Google Scholar). Direct evidence that hTopo IIIα possesses the relevant decatenase activity, however, is currently lacking.RecQ helicases and type IA topoisomerases also cooperate to resolve converging replication forks. EcTop3 together with RecQ and the single-stranded DNA-binding protein SSB catalyzes the unlinking of late replication intermediates in vitro (13Suski C. Marians K.J. Mol. Cell. 2008; 30: 779-789Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). EcTop3 also catalyzes efficient decatenation of daughter DNA molecules during oriC and pBR322 replication in vitro (14Hiasa H. DiGate R.J. Marians K.J. J. Biol. Chem. 1994; 269: 2093-2099Abstract Full Text PDF PubMed Google Scholar). Although similar assays have not been performed within eukaryotes, several lines of evidence suggest that eukaryotic topoisomerase III functions in a similar role. First, Schizosaccharomyces pombe Top3 is required for normal chromosome segregation (15Goodwin A. Wang S.W. Toda T. Norbury C. Hickson I.D. Nucleic Acids Res. 1999; 27: 4050-4058Crossref PubMed Scopus (98) Google Scholar). Second, Topo IIIα depletion in chicken DT40 cells causes accumulation of metaphase cells with chromosome gaps and breaks (16Seki M. Nakagawa T. Seki T. Kato G. Tada S. Takahashi Y. Yoshimura A. Kobayashi T. Aoki A. Otsuki M. Habermann F.A. Tanabe H. Ishii Y. Enomoto T. Mol. Cell. Biol. 2006; 26: 6299-6307Crossref PubMed Scopus (42) Google Scholar). Finally, hTopo IIIα localizes to ultrafine anaphase DNA bridges in a BLM-dependent manner (17Chan K.L. North P.S. Hickson I.D. EMBO J. 2007; 26: 3397-3409Crossref PubMed Scopus (303) Google Scholar). In each of these cases, the failure of Topo III to decatenate and thereby resolve converging replication forks could lead to interlinked sister chromatids after replication and improper sister chromatid disjunction in mitosis.In eukaryotes, Topo III functions in concert with RMI (RecQ-mediated genomic instability) proteins (18Singh T.R. Ali A.M. Busygina V. Raynard S. Fan Q. Du C.H. Andreassen P.R. Sung P. Meetei A.R. Genes Dev. 2008; 22: 2856-2868Crossref PubMed Scopus (165) Google Scholar, 19Chang M. Bellaoui M. Zhang C. Desai R. Morozov P. Delgado-Cruzata L. Rothstein R. Freyer G.A. Boone C. Brown G.W. EMBO J. 2005; 24: 2024-2033Crossref PubMed Scopus (135) Google Scholar, 20Mullen J.R. Nallaseth F.S. Lan Y.Q. Slagle C.E. Brill S.J. Mol. Cell. Biol. 2005; 25: 4476-4487Crossref PubMed Scopus (131) Google Scholar, 21Raynard S. Bussen W. Sung P. J. Biol. Chem. 2006; 281: 13861-13864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar, 23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar, 24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar). In Saccharomyces cerevisiae, deletion of the gene encoding Rmi1 results in phenotypes similar to top3Δ, including sensitivity to DNA-damaging agents, and hyper-recombination (19Chang M. Bellaoui M. Zhang C. Desai R. Morozov P. Delgado-Cruzata L. Rothstein R. Freyer G.A. Boone C. Brown G.W. EMBO J. 2005; 24: 2024-2033Crossref PubMed Scopus (135) Google Scholar, 20Mullen J.R. Nallaseth F.S. Lan Y.Q. Slagle C.E. Brill S.J. Mol. Cell. Biol. 2005; 25: 4476-4487Crossref PubMed Scopus (131) Google Scholar), consistent with Rmi1 and Top3 functioning in the same pathway. In humans, RMI1 binds to hTopo IIIα via its conserved N-terminal domain (21Raynard S. Bussen W. Sung P. J. Biol. Chem. 2006; 281: 13861-13864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar), and this interaction appears to be important for hTopo IIIα stability in vivo (23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar). In vitro, RMI1 stimulates DHJ dissolution by hTopo IIIα and BLM (21Raynard S. Bussen W. Sung P. J. Biol. Chem. 2006; 281: 13861-13864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar). Although other type IA topoisomerases can function in DHJ dissolution in vitro, stimulation of dissolution by RMI1 specifically requires hTopo IIIα (22Wu L. Bachrati C.Z. Ou J. Xu C. Yin J. Chang M. Wang W. Li L. Brown G.W. Hickson I.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 4068-4073Crossref PubMed Scopus (217) Google Scholar). RMI1 associates with a second RMI protein, RMI2, via the OB-fold domain at the C terminus to form a heterodimeric RMI complex (24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar). The RMI complex is required for the phosphorylation of BLM in mitotic cells and for the recruitment of BLM to nuclear foci in response to DNA damage (18Singh T.R. Ali A.M. Busygina V. Raynard S. Fan Q. Du C.H. Andreassen P.R. Sung P. Meetei A.R. Genes Dev. 2008; 22: 2856-2868Crossref PubMed Scopus (165) Google Scholar, 23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar, 24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar). Moreover, cells that are depleted of the RMI complex show an elevated level of SCE (23Yin J. Sobeck A. Xu C. Meetei A.R. Hoatlin M. Li L. Wang W. EMBO J. 2005; 24: 1465-1476Crossref PubMed Scopus (148) Google Scholar, 24Xu D. Guo R. Sobeck A. Bachrati C.Z. Yang J. Enomoto T. Brown G.W. Hoatlin M.E. Hickson I.D. Wang W. Genes. Dev. 2008; 22: 2843-2855Crossref PubMed Scopus (152) Google Scholar), the hallmark of BS cells, indicating that the RMI complex is critical for BLM function. Thus, the complex of BLM-hTopo IIIα-RMI1-RMI2 likely represents the functional unit in higher eukaryotes and is termed the BLM core complex (25Liu Y. West S.C. Genes. Dev. 2008; 22: 2737-2742Crossref PubMed Scopus (27) Google Scholar).To define the biochemical role of hTopo IIIα in DHJ dissolution and in the resolution of converging replication forks, we have generated a catenane of single-stranded DNA circles to mimic the proposed target of hTopo IIIα in the final step of these reactions. Using an improved procedure for the expression and purification of hTopo IIIα, we demonstrate that hTopo IIIα acts as a single-stranded DNA decatenase in DHJ dissolution and that this decatenase activity is specifically stimulated by RMI1. Surprisingly, we also find that BLM stimulates decatenation, and that BLM and RMI1 together synergistically stimulate decatenation by hTopo IIIα. We propose that hTopo IIIα is the cellular decatenase that functions in dissolution of DHJs and resolution of converging replication forks and that the optimal decatenase activity depends on the formation of a complex with RMI1 as well as BLM helicase activity.

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