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Structural and Mechanistic Insight Into Holliday-Junction Dissolution By ARTICLES Structural and mechanistic insight into Holliday-junction dissolution by Topoisomerase IIIα and RMI1 Nicolas Bocquet1, Anna H Bizard2, Wassim Abdulrahman1, Nicolai B Larsen2, Mahamadou Faty1, Simone Cavadini1, Richard D Bunker1, Stephen C Kowalczykowski3, Petr Cejka3,4, Ian D Hickson2 & Nicolas H Thomä1 Repair of DNA double-strand breaks via homologous recombination can produce double Holliday junctions (dHJs) that require enzymatic separation. Topoisomerase IIIa (TopIIIa) together with RMI1 disentangles the final hemicatenane intermediate obtained once dHJs have converged. How binding of RMI1 to TopIIIa 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 TopIIIa complexed to the first oligonucleotide-binding domain (OB fold) of RMI1. TopIIIa assumes a toroidal type 1A topoisomerase fold. RMI1 attaches to the edge of the gate in TopIIIa through which DNA passes. RMI1 projects a 23-residue loop into the TopIIIa gate, thereby influencing the dynamics of its opening and closing. Our results provide a mechanistic rationale for how RMI1 stabilizes TopIIIa-gate opening to enable dissolution and illustrate how binding partners modulate topoisomerase function. Homologous recombination (HR) is a central pathway for the repair of complex in humans19–21. Together, the RecQ helicase, Top3 (TopIIIα) DNA double-strand breaks and single-stranded gaps and is required and Rmi1 (RMI1) proteins form the minimal dHJ dissolvasome. for the maintenance and restarting of stalled replication forks1,2. Loss or mutation of any component of this complex results in HR uses a homologous DNA sequence as a template for the repair genomic instability18,22,23. of the damaged DNA strand. In canonical HR, after DNA synthesis The dHJ dissolution involves first a branch-migration step that and ligation, a dHJ intermediate is produced, which typically requires a helicase (BLM or Sgs1)24 to make the junctions migrate interconnects two sister chromatids3,4. These dHJ intermediates toward each other and a topoisomerase (TopIIIα or Top3) to relieve 5–7 25–27 Nature America, Inc. All rights reserved. Inc. Nature America, are processed, in either a crossover or a noncrossover fashion to positive supercoiling between the migrating junctions . This 4 produce recombinant products. Several nucleases are able to cleave process collapses the two HJs into a hemicatenane, and it is followed the symmetrical Holliday junction to yield an equal number of cross­ by a decatenation step that unhooks the hemicatenane intermediate 25,26 © 201 over or noncrossover products. Mitotic crossovers, however, carry into two separate DNA duplexes . the risk of loss of heterozygosity (LOH), a known driver of oncogenic TopIIIα is a member of the type 1A family of topoisomerases, which transformation8,9. In mitotic cells, LOH appears to be rare, contrib- form a toroidal structure and introduce a transient single-stranded uting to only 2.5% of HR-mediated double-strand-breakage repair nick in one DNA strand (cut strand or C strand), thus allowing a events10. Alternative pathways must therefore exist to bias HR events second single-stranded DNA (ssDNA) strand to be transferred revers- toward a noncrossover outcome. ibly through the nick (transfer strand or T strand). When T and The DNA molecules within the dHJ intermediate are topologically C strands belong to separate duplexes, the outcome is decatenation linked. The disjunction of the entangled chromosomes can be accom- or catenation (or, alternatively, hemicatenation if only one strand plished through the duplex-unwinding activity of a specific helicase is moved) and in the case of an HJ substrate, dHJ dissolution or coupled to DNA-strand unlinking by a type 1A topoisomerase. dHJ formation. Should the T and C strands belong to the same, plecto­ The interplay between RecQ helicases and topoisomerases, which is nemically linked duplex, relaxation then occurs. The in-and-out conserved in all kingdoms of life, exclusively generates noncrossover movement of a DNA single strand (T strand) is made possible by a products11 via a process termed dHJ dissolution. In humans, TopIIIα gate of intertwined β-sheets allowing reversible opening and clos- acts with BLM11, a RecQ helicase that is mutated in the cancer- ing of the torus28. In this context, the C strand is cut in a reversible predisposition disorder Bloom’s syndrome. The yeast counterparts of transesterification process giving rise to a 5′-phosphotyrosine ssDNA these proteins are Top3 topoisomerase and Sgs1 helicase12–14. Although intermediate, with the T strand being passed through the nick29. Top3 Sgs1 (BLM) and Top3 (TopIIIα) are required to process various and TopIIIα, in isolation, are moderately effective in relaxing negatively topologically linked substrates15,16, they act in concert with an addi- supercoiled plasmids, but they do possess modest ssDNA-decatenase tional essential component: Rmi1 in yeast17,18 and the RMI1–RMI2 activity30. In yeast, as in humans21, efficient decatenation requires 1Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. 2Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark. 3Department of Microbiology and Molecular Genetics, University of California, Davis, Davis, California, USA. 4Present address: Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland. Correspondence should be addressed to N.H.T. ([email protected]). Received 15 March 2013; accepted 17 January 2014; published online 9 February 2014; doi:10.1038/nsmb.2775 NATURE STRUCTURAL & MOLECULAR BIOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES Figure 1 Overall architecture and TopIIIα–RMI1 a 1 20 194 261 321 447 518 637 655 694 1,001 binding interface. (a) Domain organization Toprim IV II III II IV of the human TopIIIα–RMI1 complex. TopIIIα C4 Zn C terminus (b) Overall architecture of the crystallized 1 59 94 134 216 625 DUF1767 OB OB complex showing domains I, toprim domain RMI1 N C Linker (cyan); II, gate domain with the two topo-fold Insertion loop domains (green); III, catalytic 5Y cap (dark blue); and IV, noncatalytic CAP domain (gray). b c RMI1 is shown in red. The catalytic metal is α12 L302 RMI1 represented by a red sphere. (c) Hydrophobic β4 α12 L177 zipper between RMI1 residues originating from Domain II N terminus V298 β9 L181 OBN and residues originating from the α12 262–321 & 448–518 L297 and β10–α12 gate of TopIIIα. (d) As in c but V179 β10 Y151 depicting the corresponding hydrogen-bonding L299 M149 RMI1 insertion RMI1 A295 network. (e) Interaction between the RMI1- β15 β2 loop TopIIIα loop segment and the hydrophobic patch of the C terminus L290 M134 M136 TopIIIα gate (helix α19) (near proposed pivot Domain III α13 V95 β1 point shown in Fig. 5). RMI1 P98 is engaged 322–447 α11 in hydrophobic stacking with TopIIIα residue Domain IV d F94; neighboring RMI1 Y100 stacks with α19 195–261 E305 K78 RMI1 1 TopIIIα F262. (f) Similar to e, highlighting Domain I β 21–194 α12 the corresponding hydrogen-bonding and salt- R176 bridge network. RMI1 Q113 is at the center of C terminus Y151 4 2+ 2+ β a hydrogen-bonding network binding TopIIIα N terminus Mg or Ca E254, H530 and K529 while ionic interaction β5A TopIIIα between RMI1 E119 and TopIIIα R338 locks the RMI1 insertion loop in position. TopIIIα β3 R287 90° the presence of Rmi1, which also serves to e F94 αI αc 25 inhibit the relaxation activity . Biochemical OB1 DUF1767 I211 β15 analysis of this regulatory behavior RMI P98 β5a insertion Y100 led to the discovery that Rmi1 increases loop β3 the steady-state level of the so-called open V116 V263 2 β A118 F262 complex of Top3 and suggested a model in 4 β α19 β1 which Rmi1 stabilizes the open conforma- α13 L526 α11 25 αc tion of the topoisomerase gate . The struc- A525 A521 I tural basis by which RMI1 (Rmi1) modulates α f Nature America, Inc. All rights reserved. Inc. Nature America, α gating of strand passage by TopIII β15 4 90° (Top3) and bestows efficient decatenation E265 αI functionality that allows dHJ dissolution has RMI1 insertion loop RMI Y100 © 201 remained elusive. insertion α11 We set out to define how RMI1 (Rmi1) loop E119 modulates TopIIIα (Top3) function by using V116 R255 R338 E254 structural and biochemical approaches. Q113 A118 We show that RMI1 modulates the dynamics H530 α13 D522 of the topoisomerase action through a loop α19 K529 lining the topoisomerase gate. The TopIIIα1–753 and RMI11–219 constructs used in crystallization, RESULTS when tested together with BLM, remained active in dHJ dissolution Construct design of the TopIIIa–RMI1 complex (Supplementary Fig. 4a), thus suggesting that these boundaries are Human TopIIIα and yeast Top3 carry distinct sequence signatures sufficient for dissolution. We solved the structure of Mg2+-bound that distinguish them from their prokaryotic Top3 counterparts TopIIIα in complex with RMI1 (TopIIIα–Mg2+–RMI1) by molecu- (Supplementary Figs. 1 and 2). In the absence of structural informa- lar replacement and refined it to a maximal resolution of 2.85 Å. tion, it is unclear to what extent the eukaryotic TopIIIα (Top3) decate- The model has good agreement with the diffraction data (Table 1) nases have diverged from their prokaryotic counterparts and how RMI1 and excellent validation statistics. We also determined the structure modulates topoisomerase function by promoting decatenase activity. of a crystal grown in the presence of Ca2+, TopIIIα–Ca2+–RMI1, at In an effort to address these questions, we determined the structure 3.25-Å resolution, and it has similar validation statistics. The domain of the TopIIIα–RMI1 complex. We obtained crystals of the human boundaries for the human TopIIIα–RMI1 (OBN) complex used for TopIIIα–RMI1 complex after 4–5 d for human TopIIIα (residues crystallization are equivalent to those of full-length Saccharomyces 1–753 after deletion of residues 754–1001) in complex with human cerevisiae Top3–Rmi1.
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