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

  • Jay Yang
    Affiliations
    Department of Biochemistry and Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada and
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  • Csanad Z. Bachrati
    Affiliations
    Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom
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  • Jiongwen Ou
    Affiliations
    Department of Biochemistry and Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada and
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  • Ian D. Hickson
    Affiliations
    Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom
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  • Grant W. Brown
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, University of Toronto, Donnelly CCBR, Rm. 1230, 160 College St., Toronto, Ontario M5S 3E1, Canada. Tel.: 416-946-5733; Fax: 416-978-8548;
    Affiliations
    Department of Biochemistry and Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada and
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  • Author Footnotes
    * This work was supported by the Canadian Institute of Health Research Grant MOP-79368 (to G. W. B.), by Cancer Research United Kingdom, and the Bloom Syndrome Foundation (to I. D. H. and C. Z. B.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. 1–6.
Open AccessPublished:May 05, 2010DOI:https://doi.org/10.1074/jbc.M110.123216
      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.

      Introduction

      Topoisomerases are ubiquitous enzymes conserved from bacteria to humans. Their roles in modulating DNA topology in replication, transcription, and other cellular processes (
      • Champoux J.J.
      ,
      • Wang J.C.
      ) 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 (
      • Champoux J.J.
      ,
      • Wang J.C.
      ). 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 (
      • Champoux J.J.
      ,
      • Wang J.C.
      ). 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
      The abbreviations used are: Topo
      topoisomerase
      DHJ
      double Holliday junction
      DTT
      dithiothreitol
      BSA
      bovine serum albumin
      hTopo
      human topoisomerase
      BS
      Bloom syndrome
      SCE
      sister chromatid exchange
      oligo
      oligonucleotide
      GST
      glutathione 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 (
      • Champoux J.J.
      ). 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 (
      • Champoux J.J.
      ). 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 (
      • Mankouri H.W.
      • Hickson I.D.
      ). RecQ helicases are a highly conserved family of DNA helicases that are required for the maintenance of genome integrity (
      • Hickson I.D.
      ). Human topoisomerase IIIα (hTopo IIIα) physically interacts with BLM, one of the five RecQ helicases in humans (
      • Wu L.
      • Davies S.L.
      • North P.S.
      • Goulaouic H.
      • Riou J.F.
      • Turley H.
      • Gatter K.C.
      • Hickson I.D.
      ,
      • Johnson 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.
      ). Biallelic mutations of BLM give rise to a clinically defined cancer predisposition disorder, Bloom syndrome (BS) (
      • German J.
      ). BS cells display signs of genome instability, featuring an ∼10-fold elevation in the frequency of sister chromatid exchanges (SCE) (
      • Chaganti R.S.
      • Schonberg S.
      • German J.
      ), events that arise from the processing of recombination intermediates (
      • Sonoda E.
      • Sasaki M.S.
      • Morrison C.
      • Yamaguchi-Iwai Y.
      • Takata M.
      • Takeda S.
      ). The hTopo IIIα interacting domain of BLM is required for suppression of SCE in BS cells (
      • Hu P.
      • Beresten S.F.
      • van Brabant A.J.
      • Ye T.Z.
      • Pandolfi P.P.
      • Johnson F.B.
      • Guarente L.
      • Ellis N.A.
      ), 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 (
      • Wu L.
      • Hickson I.D.
      ). 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 (
      • Wu L.
      • Hickson I.D.
      ). 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 (
      • Wu L.
      • Hickson I.D.
      ,
      • Plank J.L.
      • Wu J.
      • Hsieh T.S.
      ). 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 (
      • Suski C.
      • Marians K.J.
      ). EcTop3 also catalyzes efficient decatenation of daughter DNA molecules during oriC and pBR322 replication in vitro (
      • Hiasa H.
      • DiGate R.J.
      • Marians K.J.
      ). 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 (
      • Goodwin A.
      • Wang S.W.
      • Toda T.
      • Norbury C.
      • Hickson I.D.
      ). Second, Topo IIIα depletion in chicken DT40 cells causes accumulation of metaphase cells with chromosome gaps and breaks (
      • Seki 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.
      ). Finally, hTopo IIIα localizes to ultrafine anaphase DNA bridges in a BLM-dependent manner (
      • Chan K.L.
      • North P.S.
      • Hickson I.D.
      ). 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 (
      • Singh T.R.
      • Ali A.M.
      • Busygina V.
      • Raynard S.
      • Fan Q.
      • Du C.H.
      • Andreassen P.R.
      • Sung P.
      • Meetei A.R.
      ,
      • Chang M.
      • Bellaoui M.
      • Zhang C.
      • Desai R.
      • Morozov P.
      • Delgado-Cruzata L.
      • Rothstein R.
      • Freyer G.A.
      • Boone C.
      • Brown G.W.
      ,
      • Mullen J.R.
      • Nallaseth F.S.
      • Lan Y.Q.
      • Slagle C.E.
      • Brill S.J.
      ,
      • Raynard S.
      • Bussen W.
      • Sung P.
      ,
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ,
      • Yin J.
      • Sobeck A.
      • Xu C.
      • Meetei A.R.
      • Hoatlin M.
      • Li L.
      • Wang W.
      ,
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ). In Saccharomyces cerevisiae, deletion of the gene encoding Rmi1 results in phenotypes similar to top3Δ, including sensitivity to DNA-damaging agents, and hyper-recombination (
      • Chang M.
      • Bellaoui M.
      • Zhang C.
      • Desai R.
      • Morozov P.
      • Delgado-Cruzata L.
      • Rothstein R.
      • Freyer G.A.
      • Boone C.
      • Brown G.W.
      ,
      • Mullen J.R.
      • Nallaseth F.S.
      • Lan Y.Q.
      • Slagle C.E.
      • Brill S.J.
      ), consistent with Rmi1 and Top3 functioning in the same pathway. In humans, RMI1 binds to hTopo IIIα via its conserved N-terminal domain (
      • Raynard S.
      • Bussen W.
      • Sung P.
      ,
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ), and this interaction appears to be important for hTopo IIIα stability in vivo (
      • Yin J.
      • Sobeck A.
      • Xu C.
      • Meetei A.R.
      • Hoatlin M.
      • Li L.
      • Wang W.
      ). In vitro, RMI1 stimulates DHJ dissolution by hTopo IIIα and BLM (
      • Raynard S.
      • Bussen W.
      • Sung P.
      ,
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ). Although other type IA topoisomerases can function in DHJ dissolution in vitro, stimulation of dissolution by RMI1 specifically requires hTopo IIIα (
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ). RMI1 associates with a second RMI protein, RMI2, via the OB-fold domain at the C terminus to form a heterodimeric RMI complex (
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ). 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 (
      • Singh T.R.
      • Ali A.M.
      • Busygina V.
      • Raynard S.
      • Fan Q.
      • Du C.H.
      • Andreassen P.R.
      • Sung P.
      • Meetei A.R.
      ,
      • Yin J.
      • Sobeck A.
      • Xu C.
      • Meetei A.R.
      • Hoatlin M.
      • Li L.
      • Wang W.
      ,
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ). Moreover, cells that are depleted of the RMI complex show an elevated level of SCE (
      • Yin J.
      • Sobeck A.
      • Xu C.
      • Meetei A.R.
      • Hoatlin M.
      • Li L.
      • Wang W.
      ,
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ), 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 (
      • Liu Y.
      • West S.C.
      ).
      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.

      EXPERIMENTAL PROCEDURES

      Protein Expression and Purification

      hTopo IIIα cDNA was subcloned from pET29H2-hTopo III (
      • Goulaouic H.
      • Roulon T.
      • Flamand O.
      • Grondard L.
      • Lavelle F.
      • Riou J.F.
      ) into pRS424-Gal-GST (
      • Bylund G.O.
      • Majka J.
      • Burgers P.M.
      ) to generate pRS424Gal-GST-hTopoIIIα. This plasmid was transformed along with plasmid pMTL4 (
      • Puig O.
      • Caspary F.
      • Rigaut G.
      • Rutz B.
      • Bouveret E.
      • Bragado-Nilsson E.
      • Wilm M.
      • Séraphin B.
      ) into the protease-deficient S. cerevisiae strain BJ2168 (MATa ura3-52 trp1-289 leu2-3 prb1-1122 prc1-407 pep4-3). Methods for cell growth, protein induction, and extract preparation were essentially the same as described previously (
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ), except that the cells were lysed in HEPES100 lysis buffer (50 mm HEPES-NaOH (pH 7.5), 100 mm NaCl, 10% glycerol, 0.1% Tween 20, 1 mm EDTA, 1 mm EGTA, 3 mm DTT, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 10 mm sodium bisulfite, 10 μg/ml leupeptin, and 1 μg/ml pepstatin A). To separate DNA from soluble proteins, the extract was treated with 0.45% polymin-P and 600 mm ammonium sulfate at 4 °C for 10 min, followed by centrifugation at 20,000 rpm for 45 min. The soluble fraction was then precipitated with 2 m ammonium sulfate, and the precipitate was resuspended and dialyzed overnight against HEPES150 lysis buffer (which has the same composition as HEPES100, except with 150 mm NaCl). The soluble fraction was loaded onto a 5-ml heparin-Sepharose column (HiTrap heparin, GE Healthcare) using fast protein liquid chromatography (GE Healthcare), and the column was washed with HEPES150 for 10 column volumes prior to elution with a NaCl gradient from 150 mm to 1 m over 10 column volumes. Fractions containing hTopo IIIα were identified by immunoblotting and fractionated on a 1-ml glutathione-Sepharose column (GSTrap 4B; GE Healthcare). The column was washed with HEPES545 (545 mm NaCl) for 10 column volumes before the bound material was eluted by the addition of HEPES545 containing 20 mm glutathione.
      RMI1 wild type and mutants were expressed in S. cerevisiae BJ2168 strains lacking SGS1 and TOP3 (MATa sgs1Δ::kanR top3Δ::natR ura3-52 trp1-289 leu2-3 prb1-1122 prc1-407 pep4-3) and were purified as described previously (
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ). Bacterial purified hTopo IIIα was a gift from J.-F. Riou and H. Goulaouic (both of Aventis Pharma, France). BLM was expressed and purified as described previously (
      • Karow J.K.
      • Chakraverty R.K.
      • Hickson I.D.
      ). RMI2 was a gift from Weidong Wang (National Institutes of Health, Baltimore). EcTop1 (M0301S) and BSA (B9001S) were purchased from New England Biolabs. hTopl was a gift from Tom Melendy (State University at Buffalo).

      Generation of RMI1 Mutants

      RMI1 mutants, LLTD and K166A were generated from p426Gal1-hRMI1-TAP by QuikChange mutagenesis. The oligonucleotides used for this study were purchased from Integrated DNA Technologies. Their sequences are in supplemental Table 1. All mutants were confirmed by sequencing.

      Generation of Single-stranded Catenanes

      The single strand catenane substrate was constructed as described previously (
      • Bucka A.
      • Stasiak A.
      ), with several modifications. Briefly, oligos C1 and C2 were annealed in the presence of oligo B1 to form the catenane. Control reactions that contained only oligo C2 also contained oligo B2 to allow circularization of C2 to occur efficiently. Single-stranded oligonucleotides were radiolabeled individually with [γ-32P]ATP (BLU502A; PerkinElmer Life Sciences) using T4 polynucleotide kinase (M0201S, New England Biolabs) prior to use. After ligation of the catenated oligos, the single-stranded catenane was purified by electrophoresis on a 12% polyacrylamide gel containing 8 m urea in TBE buffer for 75 min at 175 V, followed by staining with SYBR Green I (S-7563; Invitrogen) for 30 min. Bands containing the single-stranded catenanes were identified and excised under UV light. Catenanes were recovered by electroelution into dialysis bags in 0.25× TBE buffer for 60 min at 120 V. The eluates were concentrated using spin filters (BIOMAX-10K NMWL, Millipore). The final product appeared as a double band on 12% denaturing polyacrylamide gels because there was likely a mixture of full-length and truncated oligos in the starting material due to the long runs of dT residues. The sequences of the DNA oligonucleotides used for the generation of single-stranded catenanes are in supplemental Table 2.

      Single-stranded DNA Decatenation Assay

      Purified single-stranded catenane (400 fmol) was incubated with the indicated proteins in 15 μl of reaction buffer containing 50 mm Tris-HCl (pH 7.5), 40 mm NaCl, 5 mm MgCl2, 1 mm DTT, and 0.1 mg/ml BSA at 37 °C for 30 min, unless indicated otherwise. Reactions were stopped by the addition of 1% SDS and 20 mm EDTA. Samples were deproteinized with 1 mg/ml proteinase K at 50 °C for 1 h before being subjected to 12% polyacrylamide + 8 m urea gel electrophoresis in TBE buffer at 175 V for 75 min. Gels were fixed in 10% MeOH, 5% acetic acid for 30 min and equilibrated in 1% glycerol, 10% acetic acid for another 30 min prior to drying. Gels were then exposed to a storage phosphor screen and subsequently analyzed with a Typhoon scanner (GE Healthcare) and ImageJ software. For reactions containing BLM, 5 mm ATP was added to initiate the reactions, unless indicated otherwise.

      DNA Relaxation Assay

      pBR322 (21 fmol) (N3033S, New England Biolabs) was incubated with the indicated proteins in 15 μl of reaction buffer containing 50 mm Tris-HCl (pH 7.5), 40 mm NaCl, 5 mm MgCl2, 1 mm DTT, and 0.1 mg/ml BSA at 37 °C for 30 min. Reactions were stopped by the addition of 3 μl of Stop Buffer containing 2.5% SDS, 25 mm EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol, and 15% glycerol. Samples were subjected to 1% agarose gel electrophoresis in TAE buffer with buffer re-circularization at 70 V for 16 h. The gels were stained with SYBR Green I (S-7563; Invitrogen) for 30 min and visualized under UV light.

      Peptide Binding Assay

      Peptide arrays were prepared by the CRUK Peptide Synthesis Laboratory. Peptides were synthesized on a Multipep Synthesizer (Intavis Bioanalytical Instruments, Cologne, Germany) on derivatized cellulose (amino-Peg500 UC540, Intavis). After synthesis and deprotection, the membranes were washed with dichloromethane, N-methylpyrrolidone, and then ethanol. Binding of hTopo IIIα to the immobilized peptides was analyzed by the far-Western technique using hTopo IIIα as probe and detecting with the D6 anti-hTopo IIIα antibody, as described previously (
      • Wu L.
      • Davies S.L.
      • North P.S.
      • Goulaouic H.
      • Riou J.F.
      • Turley H.
      • Gatter K.C.
      • Hickson I.D.
      ).

      Co-immunoprecipitation Assay

      S. cerevisiae strain BJ2168 expressing GST-hTopo IIIα and wild type or mutant forms of RMI1 were harvested, washed and resuspended in lysis buffer containing 100 mm Tris-HCl (pH 8.3), 100 mm NaCl, 10% glycerol, 0.1% Tween 20, 1 mm EDTA, 5 mm sodium pyrophosphate, 0.5 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, and 1 μg/ml pepstatin A. An equal volume of glass beads was added, and the cells were lysed by vortexing at 4 °C for 10 min. Extracts were clarified by centrifuging at 8000 rpm at 0 °C for 15 min and then incubated with glutathione-Sepharose 4B (17-0757-01, GE Healthcare) at 4 °C for 2 h. Immunoprecipitates were washed five times with wash buffer containing 50 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 0.1% Tween 20, 10 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 50 mm NaF, 10 mm NaHSO4, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, and 1 μg/ml pepstatin A, prior to being eluted by the addition of SDS-PAGE sample buffer. Proteins were resolved on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis with anti-GST (sc-138; Santa Cruz Biotechnology) and anti-TAP (peroxidase-anti-peroxidase soluble complex; Sigma) antibodies.

      DHJ Dissolution Assay

      The DHJ dissolution assay was performed as described previously (
      • Bachrati C.Z.
      • Hickson I.D.
      ).

      RESULTS

      Expression and Purification of Recombinant hTopo IIIα

      Because hTopo IIIα does not display a strong biochemical activity when purified from Escherichia coli, we developed a procedure for the expression and purification of hTopo IIIα in yeast (supplemental Fig. 1A). hTopo IIIα tagged at the N terminus with GST was expressed in S. cerevisiae using a galactose-inducible expression system (
      • Burgers P.M.
      ). Using affinity chromatography together with two rounds of conventional column chromatography, we purified hTopo IIIα to near homogeneity (supplemental Fig. 1B). The purified hTopo IIIα was catalytically active as evidenced by its ability to relax negatively supercoiled pBR322 plasmid DNA (Fig. 1A). Furthermore, the DNA relaxation activity of hTopo IIIα was dependent on Mg2+ (Fig. 1B), as reported previously (
      • Goulaouic H.
      • Roulon T.
      • Flamand O.
      • Grondard L.
      • Lavelle F.
      • Riou J.F.
      ), indicating that the preparation was not contaminated by detectable amounts of Top1.
      Figure thumbnail gr1
      FIGURE 1Relaxation of negatively supercoiled DNA by hTopo IIIα. A, relaxation reactions containing increasing concentrations of hTopo IIIα (10 nm, lane 2; 20 nm, lane 3; and 40 nm, lane 4). scDNA, supercoiled DNA; rcDNA, relaxed, covalently closed DNA. B, relaxation reactions containing increasing concentrations of human topoisomerase IIIα (7.5 nm, lanes 2 and 6; 15 nm, lanes 3 and 7) or human topoisomerase I (5 nm) in the presence or absence of magnesium. C, relaxation reactions containing hTopo IIIα (10 nm, lanes 2–5 and 7–9), increasing concentrations of RMI1 (100 nm, lane 3; 200 nm, lane 4; and 400 nm, lanes 5 and 6), or increasing concentrations of BSA (100 nm, lane 7; 200 nm, lane 8; and 400 nm, lanes 9 and 10). The percent of relaxed, covalently closed DNA relative to supercoiled DNA is indicated. D, relaxation reactions containing increasing concentrations of EcTop1 (4–64 nm; lanes 2–6). E, relaxation reactions containing EcTop1 (8 nm, lanes 2–5 and 7–9), increasing concentrations of RMI1 (100 nm, lane 3; 200 nm, lane 4; and 400 nm, lanes 5 and 6), or increasing concentrations of BSA (100 nm, lane 7; 200 nm, lane 8; and 400 nm, lanes 9 and 10).

      RMI1 Specifically Stimulates the DNA Relaxation Activity of hTopo IIIα

      It has been proposed that RMI1 promotes BLM-hTopo IIIα-mediated DHJ dissolution by influencing the activity of hTopo IIIα (
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ). To test this directly, we expressed TAP-tagged RMI1 in an S. cerevisiae strain lacking SGS1 and TOP3 to prevent potential Top3 contamination. RMI1 was purified to near-homogeneity (supplemental Fig. 1, C and D) (
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ), and we measured the effect of RMI1 on hTopo IIIα-mediated DNA relaxation. At the low topoisomerase concentrations used here, hTopo IIIα exhibited a weak DNA relaxation activity (Fig. 1C). Relaxation activity was stimulated by the addition of increasing amounts of RMI1. hTopo IIIα relaxation activity was not stimulated when it was incubated with the same concentrations of BSA suggesting that the stimulation, despite requiring a large molar excess of RMI1, was specific (Fig. 1C).
      Other type IA topoisomerases, such as EcTop1, are able to function with BLM in DHJ dissolution (
      • Wu L.
      • Hickson I.D.
      ). However, RMI1 does not stimulate the BLM-EcTop1-mediated DHJ dissolution (
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ). Consistent with this observation, the relaxation activity of EcTop1 was not stimulated by RMI1 (Fig. 1, D and E). This further suggests that the stimulation of hTopo IIIα by RMI1 is specific, and indicates that the stimulation of hTopo IIIα in both relaxation and DHJ dissolution requires the cognate topoisomerase-RMI1 pair.
      RMI2, a member of the BLM core complex, is critical for hTopo IIIα and RMI1 protein function in vivo (
      • Singh T.R.
      • Ali A.M.
      • Busygina V.
      • Raynard S.
      • Fan Q.
      • Du C.H.
      • Andreassen P.R.
      • Sung P.
      • Meetei A.R.
      ,
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ). We therefore measured the effect of RMI2 on hTopo IIIα-mediated DNA relaxation. We found that RMI2 did not enhance hTopo IIIα relaxation activity on its own, nor did it stimulate hTopo IIIα relaxation activity in concert with RMI1 (supplemental Fig. 2A).

      Generation of Single-stranded Catenanes

      Although relaxation assays are typically used to measure topoisomerase activity in vitro, it is unlikely that DNA relaxation is the biologically relevant activity of hTopo IIIα. Rather, it has been proposed that the role of hTopo IIIα in resolving recombination intermediates is to facilitate the resolution of hemicatenanes that result from the BLM helicase-mediated, convergent migration of two Holliday junctions (
      • Wu L.
      • Hickson I.D.
      ). Hemicatenanes, which contain topologically linked DNA single strands, could be resolved by a single strand decatenase activity of hTopo IIIα, although this activity has not been identified directly. We generated single-stranded DNA catenanes consisting of a 60-nucleotide oligomer (circle 1) and a 40-nucleotide oligomer (circle 2) to mimic the hemicatenanes that hTopo IIIα is proposed to resolve in DHJ dissolution (Fig. 2A). Each oligonucleotide contained a 12-bp complementary region that was flanked by flexible poly(dT) linkers to allow the formation of a mixture of singly linked and doubly linked single-stranded catenanes (
      • Bucka A.
      • Stasiak A.
      ). Consistent with the properties of a closed circular single-stranded DNA catenane, the final product exhibited slower mobility than the monomer circles on a denaturing polyacrylamide gel (Fig. 2B, lanes 4 and 5 versus lane 6) and was resistant to denaturant (Fig. 2B, lane 3 versus lane 6) and exonuclease digestion (Fig. 2B, lanes 6 and 12 versus lanes 3 and 9). The purified catenane is shown in Fig. 2C.
      Figure thumbnail gr2
      FIGURE 2hTopo IIIα is a single-stranded DNA decatenase. A, schematic representation of the construction of single-stranded catenanes. RT, room temperature. B, denaturing PAGE of intermediates in the catenane assembly before (nicked; lanes 1–3 and 7–9) and after (ligated; lanes 4–6 and 10–12) ligation, and before (lanes 1–6) and after (lanes 7–12) treatment with exonucleases I and III. The samples are as follows: 60-mer circle 1 oligo (C1) + Band-Aid oligo 1 (B1), 40-mer circle 2 oligo (C2) + Band-Aid oligo 2 (B2), and a mixture of C1 + C2 + B1 (SSC). The single-stranded catenanes are resistant to denaturant and exonuclease digestion. Representations of the different oligonucleotides (OO, single-stranded catenanes; O, circular 60-mer circle 1; −, linear 60-mer circle 1; O/−, circular/linear 40-mer circle 2) are shown. RT, room temperature. C, denaturing PAGE of single-stranded catenanes before (lane 3) and after (lane 4) gel purification. Circles 1 and 2 were loaded in lanes 1 and 2, respectively, as markers. D, decatenation reactions containing increasing concentrations of hTopo IIIα (3.75–60 nm, lanes 4–8) were fractionated on a denaturing polyacrylamide gel and autoradiographed. Circles 1 and 2 were loaded in lanes 1 and 2, respectively, as markers. The percent of catenated substrate converted to circular products is indicated. E, decatenation reactions containing increasing concentrations of EcTop1 (0.375–24 nm, lanes 2–8) were fractionated on a denaturing polyacrylamide gel and autoradiographed. The percent of catenated substrate converted to circular products is indicated.

      hTopo IIIα Is a Single-stranded DNA Decatenase

      We used the single-stranded DNA catenanes to test whether hTopo IIIα displays decatenase activity in vitro. When we incubated hTopo IIIα with the single-stranded catenanes, we observed an appearance of bands corresponding to the sizes of individual circles 1 and 2 and a concomitant disappearance of the catenated species (Fig. 2D), indicating that hTopo IIIα indeed possesses single-stranded DNA decatenase activity.
      Unlike type IB topoisomerases, members of the type IA DNA topoisomerase family from non-human sources are able to substitute for hTopo IIIα in BLM-catalyzed DHJ dissolution (
      • Wu L.
      • Hickson I.D.
      ). This suggests that type IA but not type IB topoisomerases are able to catalyze the relevant reaction in DHJ dissolution. We reasoned that if the relevant activity in DHJ dissolution is single-stranded DNA decatenation, then type IA but not type IB topoisomerases should catalyze this decatenation reaction. We therefore repeated the decatenation experiment with EcTop1, a type IA topoisomerase, and hTopo I, a type IB topoisomerase. We found that although EcTop1 displayed clear decatenase activity (Fig. 2E), hTopo I was unable to resolve single-stranded catenanes even at a concentration ∼5 times higher than that at which relaxation activity was detected (supplemental Fig. 3, A and B). Together, these data suggest that the hTopo IIIα activity that is relevant to DHJ dissolution by BLM and hTopo IIIα is single-stranded DNA decatenation and not supercoil relaxation.

      Characterization of hTopo IIIα-mediated Decatenation of Single-stranded Catenanes

      Next, we characterized the biochemical properties of the decatenase activity of hTopo IIIα. The decatenase activity of hTopo IIIα was maximal when the reactions were incubated at 30 °C, although it was almost completely abolished at 50 °C (supplemental Fig. 4A). The decatenase activity of hTopo IIIα decreased linearly with both increasing NaCl concentration and increasing pH. Activity was strongest at NaCl concentrations below 20 mm and at a pH of less than 6.5 (supplemental Fig. 4, B and C). hTopo IIIα decatenase activity was greatly diminished when Mg2+ concentration was reduced to less than 1.25 mm (supplemental Fig. 4D), consistent with previous reports that type IA DNA topoisomerases require Mg2+ as a cofactor for catalytic activity. Of the divalent cations tested, only Ca2+ was able to substitute for Mg2+ as a cofactor in mediating the decatenation of single-stranded catenanes by hTopo IIIα (supplemental Fig. 4, E and F).

      RMI1 Stimulates the Decatenase Activity of hTopo IIIα but Not That of EcTop1

      Because RMI1 promotes hTopo IIIα-mediated relaxation of supercoiled DNA and the BLM-hTopo IIIα-mediated dissolution of DHJs, we tested the effect of RMI1 on hTopo IIIα decatenase activity. We found that the decatenation by hTopo IIIα was stimulated by RMI1, in a concentration-dependent manner, by up to 2-fold (Fig. 3A). Furthermore, although EcTop1 decatenated single-stranded DNA, RMI1 did not promote EcTop1-mediated decatenation (Fig. 3B). Together, these data suggest that RMI1 stimulates single-stranded DNA decatenation by hTopo IIIα specifically and argues against models of stimulation in which RMI1 modifies the DNA substrate, for example, to make it more accessible to the topoisomerase.
      Figure thumbnail gr3
      FIGURE 3RMI1 stimulates hTopo IIIα decatenase activity. A, decatenation reactions containing hTopo IIIα (7.5 nm, lanes 2–5) and RMI1 (50 nm, lane 3, 100 nm, lane 4; 200 nm lanes 5 and 6) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (hTopo IIIα alone). The percent of catenated substrate converted to circular products is indicated. B, decatenation reactions containing EcTop1 (3 nm, lanes 2–4) and RMI1 (100 nm, lane 3; 200 nm lanes 4 and 5) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (EcTop1 alone). The percent of catenated substrate converted to circular products is indicated.

      Identification of hTopo IIIα-interacting Domain in RMI1

      Because specific stimulation of hTopo IIIα-mediated decatenation by RMI1 suggests that the stimulatory effect is via a direct physical interaction between the two proteins, we tested the requirement for binding of hTopo IIIα by RMI1 in stimulation of decatenation and of DHJ dissolution. We first performed an in vitro peptide binding assay (
      • Wu L.
      • Davies S.L.
      • North P.S.
      • Goulaouic H.
      • Riou J.F.
      • Turley H.
      • Gatter K.C.
      • Hickson I.D.
      ) to identify putative hTopo IIIα interacting regions in RMI1 (supplemental Fig. 5). Candidate binding regions of RMI1 were subjected to mutagenesis, and alanine substitution mutations that eliminated binding in the peptide assay were introduced into the full-length RMI1. Co-immunoprecipitation analysis was performed with the mutant proteins to test the role of each region in the hTopo IIIα-RMI1 interaction. We found that RMI1 harboring an LLTD (residues 57–60) to AAAA mutation in the conserved DUF1767 domain (RMI1-LLTD) (Fig. 4A and supplemental Fig. 5) exhibited a defect in binding to hTopo IIIα (Fig. 4B), indicating that these four amino acids contribute to the physical interaction between hTopo IIIα and RMI1. In addition to the LLTD mutation, RMI1 harboring a K166A mutation (RMI1-K166A), which was previously shown to be defective in hTopo IIIα interaction (
      • Raynard S.
      • Zhao W.
      • Bussen W.
      • Lu L.
      • Ding Y.Y.
      • Busygina V.
      • Meetei A.R.
      • Sung P.
      ), displayed reduced binding to hTopo IIIα (Fig. 4C).
      Figure thumbnail gr4
      FIGURE 4RMI1 physically interacts with hTopo IIIα via its conserved LLTD motif. A, schematic representations of RMI1 wild type (WT) and mutants. The two putative OB-folds, OB1 and OB2, and the conserved DUF1767 domain are indicated as dark and light gray boxes, respectively. Black stripes in the RMI1 mutants indicate the amino acid positions where amino acid substitution mutations were made. B and C, extracts from cells expressing the indicated epitope-tagged proteins were treated with glutathione 4B-Sepharose to precipitate GST or GST-hTopo IIIα. 1% of input (I) extracts and the precipitate (P) was fractionated by SDS-PAGE. Immunoblots were probed with anti-GST antibody to detect GST-hTopo IIIα or with peroxidase/anti-peroxidase to detect RMI1-TAP. Identities of the bands are indicated on the right. The amount of RMI1 in the precipitates relative to that in the input extracts was calculated as a percentage and is indicated below each immunoblot.

      Stimulation of DHJ Dissolution and Decatenation Depends on Physical Interaction between hTopo IIIα and RMI1

      RMI1-K166A is defective in stimulating the DHJ dissolution activity of BLM-hTopo IIIα (
      • Raynard S.
      • Zhao W.
      • Bussen W.
      • Lu L.
      • Ding Y.Y.
      • Busygina V.
      • Meetei A.R.
      • Sung P.
      ). We therefore asked whether RMI1-LLTD displays a similar defect in DHJ dissolution. Although wild type RMI1 readily stimulated DHJ dissolution at 25 nm, both RMI1-LLTD and -K166A failed to stimulate DHJ dissolution even at >300 nm (Fig. 5, A–D), confirming that the stimulatory effect of RMI1 on BLM-hTopo IIIα-mediated DHJ dissolution requires a physical interaction between hTopo IIIα and RMI1.
      Figure thumbnail gr5
      FIGURE 5hTopo IIIα binding mutants of RMI1 do not stimulate DHJ dissolution or decatenation. A–C, increasing concentrations of wild type (WT) RMI1 (A, 0.01–356 nm, lanes 2–17), RMI1-LLTD (B, 0.01–356 nm, lanes 2–17), and RMI1-K166A (C, 0.02–712 nm, lanes 2–17) were added to DHJ dissolution reactions containing BLM (15.6 nm) and hTopo IIIα (22.25 nm) at concentrations that give ∼75% dissolution in the absence of RMI1 (lane 1 in each case). D, quantification of DHJ dissolution reactions from A to C. E, decatenation reactions containing hTopo IIIα (7.5 nm, lanes 2–5, 7–9, and 11–13), wild type RMI1 (50 nm, lane 3; 100 nm, lane 4; 200 nm, lanes 5 and 6), RMI1-LLTD mutant (50 nm, lane 7; 100 nm, lane 8; 200 nm, lanes 9 and 10), and RMI1-K166A mutant (50 nm, lane 11; 100 nm, lane 12; 200 nm, lanes 13 and 14) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (hTopo IIIα alone). The percent of catenated substrate converted to circular products is indicated.
      We tested whether RMI1-LLTD and -K166A could promote single-stranded decatenation by hTopo IIIα. The decatenase activity of hTopo IIIα was not stimulated by either RMI1-LLTD or RMI1-K166A (Fig. 5E), indicating that the physical interaction between hTopo IIIα and RMI1 is critical for the RMI1 stimulation of hTopo IIIα-mediated decatenation. These data are consistent with RMI1 stimulating hTopo IIIα in DHJ dissolution, and with the stimulation being the result of RMI1 modulation of hTopo IIIα activity, rather than an indirect effect of RMI1 on DNA substrate accessibility or conformation.

      BLM, but Not RMI2, Stimulates hTopo IIIα Decatenase Activity

      Like hTopo IIIα and RMI1, BLM and RMI2 are members of the BLM core complex (
      • Liu Y.
      • West S.C.
      ). BLM promotes hTopo IIIα-mediated relaxation of negatively supercoiled DNA in vitro (
      • Wu L.
      • Hickson I.D.
      ) and is required for the proper localization of hTopo IIIα in vivo (
      • Hu P.
      • Beresten S.F.
      • van Brabant A.J.
      • Ye T.Z.
      • Pandolfi P.P.
      • Johnson F.B.
      • Guarente L.
      • Ellis N.A.
      ,
      • Chan K.L.
      • North P.S.
      • Hickson I.D.
      ). We tested the effect of BLM on decatenation by hTopo IIIα. Addition of increasing concentrations of BLM stimulated decatenation by at least 2.5-fold (Fig. 6A). The stimulation depended on the presence of ATP (Fig. 6B), suggesting that it required the catalytic activity of BLM. BLM also stimulated EcTop1-mediated decatenation, but more weakly (∼0.5-fold; Fig. 6C). Together, these data suggest that BLM stimulates decatenation in the following two ways: unwinding of the substrate results in a modest nonspecific stimulation, but further stimulation of the cognate topoisomerase is also apparent. Thus it appears that BLM alters the conformation of the substrate in addition to influencing the activity of hTopo IIIα directly.
      Figure thumbnail gr6
      FIGURE 6BLM stimulates hTopo IIIα decatenase activity. A, decatenation reactions containing hTopo IIIα (30 nm, lanes 2–4), BLM (33 nm, lane 3; 67 nm, lanes 4 and 5), and ATP (5 mm; all lanes) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (hTopo IIIα alone). The percent of catenated substrate converted to circular products is indicated. B, decatenation reactions performed as in A, but without ATP. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (hTopo IIIα alone). The percent of catenated substrate converted to circular products is indicated. C, decatenation reactions containing EcTop1 (3 nm, lanes 2–6), BLM (33 nm, lane 3; 66 nm, lanes 4, 5 and 7), and ATP (5 mm, lanes 1–4, 6 and 7) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (EcTop1 alone). The percent of catenated substrate converted to circular products is indicated.
      Addition of increasing concentrations of RMI2 had no detectable effect on hTopo IIIα decatenase activity (supplemental Fig. 2B), and it did not have an effect on the decatenase activity of the hTopo IIIα-RMI1 pair (supplemental Fig. 2C). Hence decatenation by hTopo IIIα is independent of RMI2. It is likely that RMI2 plays a role in a higher order function of the BLM core complex that is not revealed by these highly specific biochemical assays.

      BLM and RMI1 Stimulate hTopo IIIα-mediated Decatenation Synergistically

      RMI1 enhances DHJ dissolution by ∼8-fold (
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ), whereas the stimulatory effect of RMI1 on decatenation by hTopo IIIα was a relatively modest 2-fold. Therefore, it is possible that there is additional interplay between BLM, hTopo IIIα, and RMI1. When decatenation by hTopo IIIα was assayed in the presence of both BLM and RMI1, the decatenase activity increased by a striking ∼20-fold (Fig. 7A, 5th lane versus 2nd lane). Under the same conditions, BLM or RMI1 alone stimulated decatenation by only 2-fold (Fig. 7A, 3rd and 4th lanes versus 2nd lane), indicating that BLM and RMI1 stimulate the decatenase activity of hTopo IIIα synergistically. Stimulation of hTopo IIIα by BLM-RMI1 requires physical interaction between hTopo IIIα and RMI1 because the binding-defective RMI1-LLTD mutant together with BLM did not stimulate above the level seen with BLM alone (Fig. 7B, 7th lane versus 3rd lane). Thus, the physical interaction between hTopo IIIα and RMI1 is required for the optimal activity of the BLM-hTopo IIIα-RMI1 complex in both decatenation and DHJ dissolution. Furthermore, these data indicate a role for BLM function in the very last step of the reactions catalyzed by the BLM core complex.
      Figure thumbnail gr7
      FIGURE 7BLM and RMI1 synergistically stimulate hTopo IIIα decatenase activity. A, decatenation reactions containing hTopo IIIα (15 nm, lanes 2–5), BLM (33 nm, lanes 3, 5, and 6), and RMI1 (100 nm, lanes 4, 5, and 7) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. ATP (5 mm) was added to initiate the reactions. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (hTopo IIIα alone). The percent of catenated substrate converted to circular products is indicated. B, decatenation reactions containing hTopo IIIα (15 nm, lanes 2–7), BLM (33 nm, lanes 3 and 6–8), wild type RMI1 (100 nm, lanes 4, 6, and 9), and RMI1-LLTD mutant (100 nm, lanes 5, 7, and 10) as indicated were fractionated on a denaturing polyacrylamide gel and autoradiographed. ATP (5 mm) was added to initiate the reactions. Quantification of the decatenation products is presented in the histogram, normalized to the reaction in lane 2 (hTopo IIIα alone). The percent of catenated substrate converted to circular products is indicated.

      DISCUSSION

      Using 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 (
      • Suski C.
      • Marians K.J.
      ) and resolves double Holliday junctions without crossing over (
      • Wu L.
      • Hickson I.D.
      ,
      • Plank J.L.
      • Wu J.
      • Hsieh T.S.
      ). 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 RMI1

      We 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 (
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ), 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.
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ). 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.
      • Goulaouic H.
      • Roulon T.
      • Flamand O.
      • Grondard L.
      • Lavelle F.
      • Riou J.F.
      ). 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 (
      • Chen C.F.
      • Brill S.J.
      ), 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 Decatenases

      We 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 (
      • Hiasa H.
      • DiGate R.J.
      • Marians K.J.
      ,
      • Hiasa H.
      • Marians K.J.
      ). 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 (
      • Suski C.
      • Marians K.J.
      ) 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 (
      • Suski C.
      • Marians K.J.
      ). Similarly, the second component of the DHJ dissolution activity reported by Wu and Hickson (
      • Wu L.
      • Hickson I.D.
      ) 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 (
      • Champoux J.J.
      ), 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 (
      • Wu L.
      • Hickson I.D.
      ). 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 (
      • Chan K.L.
      • North P.S.
      • Hickson I.D.
      ), and depletion of hTopo IIIα causes an increase in the number of DNA bridges (
      • Temime-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.
      ).

      BLM and RMI1 Cooperate with hTopo IIIα in Decatenation

      We 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 (
      • Raynard S.
      • Zhao W.
      • Bussen W.
      • Lu L.
      • Ding Y.Y.
      • Busygina V.
      • Meetei A.R.
      • Sung P.
      ), 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 (
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ). Budding yeast Rmi1, which lacks the C-terminal region, has weak single-stranded DNA binding activity and can bind Holliday junction structures (
      • Mullen J.R.
      • Nallaseth F.S.
      • Lan Y.Q.
      • Slagle C.E.
      • Brill S.J.
      ,
      • Chen C.F.
      • Brill S.J.
      ). 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 (
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ,
      • Chen C.F.
      • Brill S.J.
      ).
      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 (
      • Chen C.F.
      • Brill S.J.
      ); knockdown of RMI1 destabilizes hTopo IIIα, whereas knockdown of BLM does not (
      • Singh T.R.
      • Ali A.M.
      • Busygina V.
      • Raynard S.
      • Fan Q.
      • Du C.H.
      • Andreassen P.R.
      • Sung P.
      • Meetei A.R.
      ,
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      ); the Top3-Rmi1 complex binds to Sgs1 (
      • Chang M.
      • Bellaoui M.
      • Zhang C.
      • Desai R.
      • Morozov P.
      • Delgado-Cruzata L.
      • Rothstein R.
      • Freyer G.A.
      • Boone C.
      • Brown G.W.
      ,
      • Chen C.F.
      • Brill S.J.
      ); deletion of rmi1 in yeast closely resembles the phenotype of top3 deletion rather than that of sgs1 deletion (
      • Chang M.
      • Bellaoui M.
      • Zhang C.
      • Desai R.
      • Morozov P.
      • Delgado-Cruzata L.
      • Rothstein R.
      • Freyer G.A.
      • Boone C.
      • Brown G.W.
      ,
      • Mullen J.R.
      • Nallaseth F.S.
      • Lan Y.Q.
      • Slagle C.E.
      • Brill S.J.
      ); stimulation of DHJ dissolution by RMI1 requires the cognate type IA topoisomerase, hTopo IIIα (
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ); and Topo IIIα and RMI1 perform meiotic functions that are independent of RecQ4a, a homolog of BLM helicase in Arabidopsis thaliana (
      • Hartung F.
      • Suer S.
      • Knoll A.
      • Wurz-Wildersinn R.
      • Puchta H.
      ,
      • Chelysheva L.
      • Vezon D.
      • Belcram K.
      • Gendrot G.
      • Grelon M.
      ).
      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 (
      • Cheok C.F.
      • Bachrati C.Z.
      • Chan K.L.
      • Ralf C.
      • Wu L.
      • Hickson I.D.
      ). 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 (
      • Bussen W.
      • Raynard S.
      • Busygina V.
      • Singh A.K.
      • Sung P.
      ). 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 (
      • Raynard S.
      • Bussen W.
      • Sung P.
      ,
      • Wu L.
      • Bachrati C.Z.
      • Ou J.
      • Xu C.
      • Yin J.
      • Chang M.
      • Wang W.
      • Li L.
      • Brown G.W.
      • Hickson I.D.
      ). 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 (
      • Suski C.
      • Marians K.J.
      ), 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 (
      • Broberg K.
      • Huynh E.
      • Schläwicke Engström K.
      • Björk J.
      • Albin M.
      • Ingvar C.
      • Olsson H.
      • Höglund M.
      ). 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.
      Figure thumbnail gr8
      FIGURE 8hTopo IIIα, BLM, and RMI1 cooperate to catalyze decatenation of Holliday junctions and Holliday junction-like structures in vivo. These structures could arise from the repair of a double-stranded break (DSB) to generate a DHJ, which is then processed into a hemicatenane before resolution. Holliday junctions could also arise from the replication fork restart pathway when a replication fork is stalled and from the maintenance of telomere stability in the alternative lengthening of telomere (ALT) pathway. The structure of converging replication forks resembles a Holliday junction and would also be decatenated by hTopo IIIα.

      Acknowledgments

      We thank Weidong Wang for RMI2 protein, Tom Melendy for hTopl, and Peter Burgers for the pRS424-Gal-GST plasmid.

      Supplementary Material

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