Repair of DNA double-strand breaks via homologous recombination can produce double Holliday junctions (dHJs) that require enzymatic separation. Topoisomerase IIIα (TopIIIα) together with RMI1 disentangles the final hemicatenane intermediate obtained once dHJs have converged. How binding of RMI1 to TopIIIα influences it to behave as a hemicatenane dissolvase, rather than as an enzyme that relaxes DNA topology, is unknown. Here, we present the crystal structure of human TopIIIα complexed to the first oligonucleotide-binding domain (OB fold) of RMI1. TopIII assumes a toroidal type 1A topoisomerase fold. RMI1 attaches to the edge of the gate in TopIIIα through which DNA passes. RMI1 projects a 23-residue loop into the TopIIIα gate, thereby influencing the dynamics of its opening and closing. Our results provide a mechanistic rationale for how RMI1 stabilizes TopIIIα-gate opening to enable dissolution and illustrate how binding partners modulate topoisomerase function.
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Petermann, E. & Helleday, T. Pathways of mammalian replication fork restart. Nat. Rev. Mol. Cell Biol. 11, 683–687 (2010).
Moynahan, M.E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).
Holliday, R. A mechanism for gene conversion in fungi. Genet. Res. 89, 285–307 (2007).
Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J. & Stahl, F.W. The double-strand-break repair model for recombination. Cell 33, 25–35 (1983).
Heyer, W.D., Ehmsen, K.T. & Solinger, J.A. Holliday junctions in the eukaryotic nucleus: resolution in sight? Trends Biochem. Sci. 28, 548–557 (2003).
Matos, J., Blanco, M.G., Maslen, S., Skehel, J.M. & West, S.C. Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis. Cell 147, 158–172 (2011).
Heyer, W.D. Recombination: Holliday junction resolution and crossover formation. Curr. Biol. 14, R56–R58 (2004).
Cavenee, W.K. et al. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305, 779–784 (1983).
Thiagalingam, S. et al. Mechanisms underlying losses of heterozygosity in human colorectal cancers. Proc. Natl. Acad. Sci. USA 98, 2698–2702 (2001).
LaRocque, J.R. et al. Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells. Proc. Natl. Acad. Sci. USA 108, 11971–11976 (2011).
Wu, L. & Hickson, I.D. The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003).
Gangloff, S., McDonald, J.P., Bendixen, C., Arthur, L. & Rothstein, R. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol. Cell. Biol. 14, 8391–8398 (1994).
Bennett, R.J., Noirot-Gros, M.F. & Wang, J.C. Interaction between yeast sgs1 helicase and DNA topoisomerase III. J. Biol. Chem. 275, 26898–26905 (2000).
Fricke, W.M., Kaliraman, V. & Brill, S.J. Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase. J. Biol. Chem. 276, 8848–8855 (2001).
Harmon, F.G., DiGate, R.J. & Kowalczykowski, S.C. RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination. Mol. Cell 3, 611–620 (1999).
Cejka, P. & Kowalczykowski, S.C. The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds Holliday junctions. J. Biol. Chem. 285, 8290–8301 (2010).
Xu, D. et al. RMI, a new OB-fold complex essential for Bloom syndrome protein to maintain genome stability. Genes Dev. 22, 2843–2855 (2008).
Mullen, J.R., Nallaseth, F.S., Lan, Y.Q., Slagle, C.E. & Brill, S.J. Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex. Mol. Cell. Biol. 25, 4476–4487 (2005).
Singh, T.R. et al. BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome. Genes Dev. 22, 2856–2868 (2008).
Raynard, S., Bussen, W. & Sung, P. A double Holliday junction dissolvasome comprising BLM, topoisomerase IIIα, and BLAP75. J. Biol. Chem. 281, 13861–13864 (2006).
Wu, L. et al. BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates. Proc. Natl. Acad. Sci. USA 103, 4068–4073 (2006).
Lai, M.S., Seki, M., Ui, A. & Enomoto, T. Rmi1, a member of the Sgs1-Top3 complex in budding yeast, contributes to sister chromatid cohesion. EMBO Rep. 8, 685–690 (2007).
Chang, M. et al. RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex. EMBO J. 24, 2024–2033 (2005).
Mankouri, H.W. & Hickson, I.D. The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific 'dissolvasome'? Trends Biochem. Sci. 32, 538–546 (2007).
Cejka, P., Plank, J.L., Dombrowski, C.C. & Kowalczykowski, S.C. Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes. Mol. Cell 47, 886–896 (2012).
Cejka, P., Plank, J.L., Bachrati, C.Z., Hickson, I.D. & Kowalczykowski, S.C. Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1–Top3. Nat. Struct. Mol. Biol. 17, 1377–1382 (2010).
Chen, C.F. & Brill, S.J. Binding and activation of DNA topoisomerase III by the Rmi1 subunit. J. Biol. Chem. 282, 28971–28979 (2007).
Corbett, K.D. & Berger, J.M. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 33, 95–118 (2004).
Tse, Y.C., Kirkegaard, K. & Wang, J.C. Covalent bonds between protein and DNA: formation of phosphotyrosine linkage between certain DNA topoisomerases and DNA. J. Biol. Chem. 255, 5560–5565 (1980).
Yang, J., Bachrati, C.Z., Ou, J., Hickson, I.D. & Brown, G.W. Human topoisomerase IIIα is a single-stranded DNA decatenase that is stimulated by BLM and RMI1. J. Biol. Chem. 285, 21426–21436 (2010).
Hoadley, K.A. et al. Structure and cellular roles of the RMI core complex from the Bloom syndrome dissolvasome. Structure 18, 1149–1158 (2010).
Wang, F. et al. Crystal structures of RMI1 and RMI2, two OB-fold regulatory subunits of the BLM complex. Structure 18, 1159–1170 (2010).
Lima, C.D., Wang, J.C. & Mondragon, A. Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I. Nature 367, 138–146 (1994).
Hansen, G., Harrenga, A., Wieland, B., Schomburg, D. & Reinemer, P. Crystal structure of full length topoisomerase I from Thermotoga maritima. J. Mol. Biol. 358, 1328–1340 (2006).
Mondragón, A. & DiGate, R. The structure of Escherichia coli DNA topoisomerase III. Structure 7, 1373–1383 (1999).
Aravind, L., Leipe, D.D. & Koonin, E.V. Toprim: a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. Nucleic Acids Res. 26, 4205–4213 (1998).
Duguet, M., Serre, M.C. & Bouthier de La Tour, C. A universal type IA topoisomerase fold. J. Mol. Biol. 359, 805–812 (2006).
Schultz, S.C., Shields, G.C. & Steitz, T.A. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees. Science 253, 1001–1007 (1991).
Brennan, R.G. & Matthews, B.W. The helix-turn-helix DNA binding motif. J. Biol. Chem. 264, 1903–1906 (1989).
Flynn, R.L. & Zou, L. Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians. Crit. Rev. Biochem. Mol. Biol. 45, 266–275 (2010).
Goulaouic, H. et al. Purification and characterization of human DNA topoisomerase IIIα. Nucleic Acids Res. 27, 2443–2450 (1999).
Changela, A., DiGate, R.J. & Mondragon, A. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. Nature 411, 1077–1081 (2001).
Changela, A., DiGate, R.J. & Mondragon, A. Structural studies of E. coli topoisomerase III-DNA complexes reveal a novel type IA topoisomerase-DNA conformational intermediate. J. Mol. Biol. 368, 105–118 (2007).
Schmidt, B.H., Burgin, A.B., Deweese, J.E., Osheroff, N. & Berger, J.M. A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases. Nature 465, 641–644 (2010).
Li, Z., Mondragon, A., Hiasa, H., Marians, K.J. & DiGate, R.J. Identification of a unique domain essential for Escherichia coli DNA topoisomerase III-catalysed decatenation of replication intermediates. Mol. Microbiol. 35, 888–895 (2000).
Feinberg, H., Lima, C.D. & Mondragon, A. Conformational changes in E. coli DNA topoisomerase I. Nat. Struct. Biol. 6, 918–922 (1999).
Xiong, B. et al. The type IA topoisomerase catalytic cycle: A normal mode analysis and molecular dynamics simulation. Proteins 71, 1984–1994 (2008).
Li, Z., Mondragon, A. & DiGate, R.J. The mechanism of type IA topoisomerase-mediated DNA topological transformations. Mol. Cell 7, 301–307 (2001).
Viard, T. & de la Tour, C.B. Type IA topoisomerases: a simple puzzle? Biochimie 89, 456–467 (2007).
Bolanos-Garcia, V.M. et al. Spatial and temporal organization of multi-protein assemblies: achieving sensitive control in information-rich cell-regulatory systems. Philos. Trans. A Math. Phys. Eng. Sci. 370, 3023–3039 (2012).
Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).
Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
Karplus, P.A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).
Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Bachrati, C.Z. & Hickson, I.D. Dissolution of double Holliday junctions by the concerted action of BLM and topoisomerase IIIα. Methods Mol. Biol. 582, 91–102 (2009).
This work was supported by the Novartis Research Foundation (to N.H.T.), grants from the Krebsforschung Schweiz (KFS-2986-08-2012) (to N.H.T.), Marie Curie Fellowship (FP7-PEOPLE-2009-IEF 253555-TOPO) (to N.B.), European Molecular Biology Organization long-term Fellowship (EMBO ALTF 693-2009) (to N.B.) and European Research Council (to N.H.T. and I.D.H.), the Nordea Foundation (to I.D.H.), The Villum Kann Rasmussen Fund (to I.D.H.) and the US National Institutes of Health (GM-41347 and GM-62653) (to S.C.K.). We are grateful to H. Stahlberg and team for use of the EM facilities at the Center for Cellular Imaging and NanoAnalytics (c-CINA). We thank S. Gasser, U. Rass, K. Shimada, J. Keusch, E. Fischer, A. Scrima, D. Hess, K. Boehm, D. Klein, H. Gut and M. Renatus for help and fruitful discussion. We would also like to thank A. Costa and colleagues at the London Research Institute for sharing results prior to publication. Part of this work was performed at beamline PXII of the Swiss Light Source at the Paul Scherrer Institute, Villigen, Switzerland.
The authors declare no competing financial interests.
Integrated supplementary information
Human TopIIIa is the upper sequence; 2. Saccharomyces cerevisiae Top3; 3. Escherichia coli Top3; 4. Escherichia coli Top1A; 5. Thermatoga maritima Top1A. The alignment has been made with T-COFFEE (http://tcoffee.vital-it.ch) and edited to show similarity percentage with a threshold set up at 3. Percentage of similarity is coloured as a gray scale, the four different levels being (from white to black): White is less than 60%, light gray is between 60 to 80%, dark gray is 80 to 99% and black is 100%.
Percentage of conservation projected on the TopIIIα-RMI1 complex. Topoisomerase conservation has been calculated from the sequences in Supplementary fig.2 and RMI1 conservation from the sequences in panel b. b) RMI1 comparison between human RMI1 and several yeast Rmi1. 1. Saccharomyces cerevisiae; 2. Zygosaccharomyces rouxii; 3. Pichia pastoris; 4. Schizosaccharomyces japonicus; 5 . Grossmannia clavigera; 6. Colletotrichum graminicola; 7. Colletotrichum higginsanum; 8. Candida dubliniensis; 9. Schizosaccharomyces pombe; 10. Homo sapiens
a) A crystal packing diagram showing the coexistence of two TopIIIα-RMI1 interfaces. To determine which interface is biologically relevant, we performed EM and single-particle analysis of negatively stained TopIIIα-RMI complex sample used for growing crystals. b) Example of a negatively stained micrograph and individual particles marked with white squares. The scale bar corresponds to 50 nm. c) Reference-free class averages (top), reprojections of the 3D reconstruction (middle row) and reprojections of the crystal structure filtered at 30 Å resolution (bottom line) as matched by cross-correlation. d) The angular distribution for the TopIIIα-RMI reconstruction mapped on a half sphere. The size of the spot is proportional to the number of raw images contributing to the class. (e) Fourier shell correlation (FSC) showing a resolution of 34 Å calculated using the 0.5 threshold criterion (dashed lines). f) Fitting of the two interfaces (ASU interface and biological interface) found in the crystal lattice in the calculated EM map. In the alternative orientation found in the ASU the orientation and location of RMI1 does not agree with the EM envelope demonstrating that the ASU interface is not the biological interface in solution. g) Different views of the fit between the crystal structure and the calculated EM map.
a) dHJs dissolution assay using the crystallized boundaries (TopIIIα-RMI1)cb at the indicated concentrations, BLM 10 nM for 10 min incubation and 30 nM for 30 min incubation. RsaI Lane was used as a migration marker for the product of dHJ dissolution. The dissolution of dHJs is proportional to the (TopIIIα-RMI1) concentration and is complete after 30 min incubation. This demonstrates that the crystallized complex is a minimal dissolvasome and defines the minimal domains belonging to a functional enzymatic core. b) Topology scheme of the crystallized complex represented as secondary structure elements with the same colour code as in Figure 1. Dashed lines represent disordered regions in the crystal. c) Crystal packing. One heterodimer is represented as surface (Green for TopIIIα and red for RMI1) and corresponds to the biological unit. The close-up shows that the RMI1 decatenation loop is not involved in any crystal contacts. d,e) 2mFo-DFc omit maps contoured at 0.5σ and final refined models obtained in the Mg2+ (d) and Ca2+ (e) conditions (see Methods) represented as ribbons; TopIIIα in green with RMI1 depicted in red.
a) Pull-down experiments; Top3 is GST N-terminally tagged and wild-type Rmi1 and rlRmi1, as used in relaxation and catenation assays, have GST N-Terminally and His C-terminally tags. Both proteins were co-expressed in High-Five cells. In the Ni2+ -NTA pull-down, Top3 is still able to interact with the rlRmi1 mutant as indicated by the presence of a Top3 band. b) M13 digest in presence of Top3-WTRmi1 and Top3-rlRmi1 complexes. Analyses were performed after 1 h and 3 h digestion. The smear shows that the initial cleavage occurring in the first step of relaxation process is not impaired by the rlRmi1 construct. c) Catenation and decatenation assays using the yeast proteins. 100 ng of pUC19 (catenation) or kDNA (decatenation) were incubated with Top3 and Sgs1 alone (lane 2 and 6), with the additon of 200 nM of GST-WTRmi1 (lane 3 and 7) or 200nM of GST-rlRmi1 (lane 4 and 8). Although GST-WTRmi1 stimulates both the pUC19 catenation (lane 2 and 3) and kDNA decatenation (lane 6 and 7) activity of Top3-Sgs1, no stimulation was observed in the presence of GST-rlRmi1 (lane 4 and 8). kDNA decatenated in the presence of Topoisomerase II (Inspiralis) was used as a migration marker for nicked and circular covalently closed (ccc) decatenation products (lane 9).
a) Uncropped gels corresponding to fig 4a .Yeast Top3 relaxation assay using pUC19 DNA to assess the effect of yeast Rmi1 wild-type (WT Rmi1) versus rlRmi1. b) Uncropped gels corresponding to fig 4c. Digest of single stranded M13 substrate with the different complexes. c) Uncropped gels corresponding to fig 4d. dHJs dissolution assay of a short synthetic dHJ junction, using the yeast Top3, Sgs1 and Rmi1. d) Uncropped gels corresponding to fig 4e. dHJs dissolution assay of a short synthetic dHJ junction using the human TopIIIα, BLM and RMI1.
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Bocquet, N., Bizard, A., Abdulrahman, W. et al. Structural and mechanistic insight into Holliday-junction dissolution by Topoisomerase IIIα and RMI1. Nat Struct Mol Biol 21, 261–268 (2014). https://doi.org/10.1038/nsmb.2775
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