Structural and mechanistic insight into Holliday-junction dissolution by Topoisomerase IIIα and RMI1

Repair of DNA double-strand breaks via homologous recombination can produce double Holiday junctions (dHJs) that require enzymatic separation. Topoisomerase III alpha (Top111 alpha) together with RMI1 disentangles the final hemicatenane intermediate obtained once dHJs have converged. How binding of RMI1 to TopIlI alpha 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 TopIII alpha complexed to the first oligonucleotide-binding domain (OB fold) of RMI1. TopIII alpha assumes a toroidal type 1A topoisomerase fold. RMI1 attaches to the edge of the gate in TopIII alpha through which DNA passes. RMI1 projects a 23-residue loop into the TopIII alpha gate, thereby influencing the dynamics of its opening and closing. Our results provide a mechanistic rationale for how RMI1 stabilizes TopIII alpha-gate opening to enable dissolution and illustrate how binding partners modulate topoisomerase function.