Structural studies of the yeast Topoisomerase III, RMI1 Sgs1 complex

Final Report Summary - TOPO (Structural studies of the yeast Topoisomerase III, RMI1 Sgs1 complex.)

The Bloom (BLM) complex composed of TopIIIα, RMI1 and 2 in higher eukaryotes in conjunction with the RecQ BLM helicase assists in the dissolution of converging Holiday junctions in a non cross-over fashion [1-3]. This specialized reaction is carried out by a typeIII topoisomerase (TopIIIα), with help of tightly associated accessory protein Rmi1 [4-7] in yeast and the RMI1-RMI2 complex in metazoan cells [2]. How Rmi1 modulates TopIII function to serve in dissolving convergent Holiday junctions has so far remained elusive. Dissolution of homologous recombination intermediates without any genetic material exchange is a way to prevent DNA deterioration and to improve genome stability. A deep understanding at a structural level would help to decypher the molecular mechanisms underlying BLM complex function. The main goal of my proposal was to crystallize and to determine the structure of the full or partial Bloom complex. This report will the two years under the Marie Curie fellowship at the FMI in the Structural Biology lab, directed by Nicolas Thomae and presents the 3.3 Å structure of the human RMI1 N-terminus bound to the TopIIIα topoisomerase. The structure shows that RMI1 attaches to the gate of the topoisomerase with help of its oligonucleotide binding OB-fold. Moreover the human TopIII is the first high resolution structure obtained for a type III mamalian topoisomerase showing its conserved overall toroïdal architecture as well as its catalytic site. This structure allows us now to imagine a plausible single strand DNA (ssDNA) passage model to catalyse the dissolution reaction in concert with RMI1.
Overall architecture. Several TopIII-RMI interfaces coexist due to the crystal packing but according to interface analysis (PISA) and already published data which involved the RMI1 OB domain as well as an insertion loop [4,9,10] inside this OB domain in the binding to TopIIIα, the real biological interface (confirmed recently by our EM experiments, data not shown) between the two proteins is shown in the figure 1.

The human TopIIIα protein retain all the structural features which have been highlighted by the E.coli Top1A [8], IIII [11,12] and the Thermotoga maritima Top1A [13] crystal structures. The crystallized protein is composed of four domains named 1 to 4 (fig1): domain 1 is the toprim (primase fold) domain carrying the acidic cluster involved in the catalytic site, domain 2 forms the so called gate made by two antiparallel topofold domains [14] and generating a ~25 Å diameter hole in the center of the protein, domains 3 and 4 share the same ‘Winged helix’ fold (also named CAP domains for catabolite activator protein) [15]. While domain 4 does not carry any catalytic residues, domain 3 possesses the conserved catalytic Tyrosine responsible for the cutting of ssDNA [12]. It is important to note that the C-terminal region including a putative Zinc finger domain potentially involved in DNA binding and predicted to be highly similar to the Thermotoga maritima Top1A C-terminus is not unambigously buildable in our structure even if some electronic densities are visible. The Nterminal RMI1 is composed of a three-α helices bundle followed by a typical OB domain fold consisting in a β-barrel as already published in [9,10]. The complex has a ‘Door-knocker’ overall shape, RMI1 contacting TopIII at the top of the gate (mainly α9 and the β9- α9 linker) via its OB domain. The so called RMI1 insertion loop (between α3 and β3) creates two other contacting zones to TopIII, the β10, β12 and β12-β13 linker in domain 2, α10 in domain 3, and α8 and 15 as well as α8-β9 linker generating a spread interface and obstructing partially the hole made by TopIII domain 2. This interface involve hydrogen bonds as well as hydrophobic interfacing residues, and one salt bridge for a total interface area of 1319 Å2.

Final Report Summary - TOPO (Structural studies of the yeast Topoisomerase III, RMI1 Sgs1 complex.)

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