Deciphering the human TopIIIα activity modulated by Rmi1 using magnetic tweezers.

Topoisomerases IA (TopoIAs) are universal and essential enzymes present in the three domains of life. Most of the Metazoa exhibit two TopoIAs-TopIIIα and TopIIIβ-assuming different roles in the cell. TopIIIα is essential for genome stability by disentangling precatenanes and hemicatenanes during DNA replication or dissolving the double Holliday junctions in recombination, with the help of several partners, such as Rmi1. However, the detail of the TopIIIα enzymatic cycle and the precise role of Rmi1 remain essentially unknown. The single-molecule approach allows to deconvolute the different early reaction steps and distinguish between intrinsic catalytic characteristics of human TopIIIα that are invariable and those that can be modulated by Rmi1. We determined that the limiting step is the TopIIIα-DNA binding, which requires a small single-stranded region. TopIIIα punctuates its catalytic cycle with long pause times to stabilize the open cleaved complex. Rmi1 helps TopIIIα trap the single-stranded DNA and therefore greatly increases the efficiency of the binding step. Rmi1 also enhances the stabilization of the open cleaved complex to favour intermolecular reactions with improved discrimination of DNA substrates. Rmi1 is therefore a crucial partner for TopIIIα in ensuring that the DNA transaction processes run smoothly in vivo.