Title: Direct observation of type IA topoisomerase conformational dynamics

Presented by: Keir C. Neuman, Ph.D.
Professor
Director Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health
Bethesda MD

Abstract

Type IA DNA topoisomerases (topo IA) are conserved among all domains of life due to their critical roles in regulating negative DNA supercoiling, DNA decatenation, and RNA processing. Topo IA share a unique ATP-independent strand-passage mechanism in which the enzyme binds and cleaves a single-stranded DNA segment, forming a protein-DNA gate through which a second strand of DNA is passed followed by religation of the cleaved DNA. Although the strand passage model of type IA topoisomerases is widely accepted, opening of the protein-DNA gate has never been directly observed.  We developed a single-molecule assay to directly measure gate opening by type IA topos.  We found that the protein-DNA gate opens by as much as 6 nm and can close against relatively large forces in excess of 16 pico-Newtons.  More recently, we discovered that the gate dynamics of bacterial type IA topos depend on the Mg2+ concentration, and that new kinetic and structural intermediates emerge at low Mg2+ concentrations. Our results demonstrate that the catalytic activity of bacterial type IA topoisomerases is regulated by the opening and closing of the protein-DNA gate and reveal an unexpected role for Mg2+ ions in modulating the structural dynamics of the enzyme. More generally, these results indicate a potential role for divalent cations in modulating protein conformational dynamics. For the human topo IA enzyme, topoisomerase 3A, we find that binding of the RMI1-2 complex, a critical regulator of cellular topoisomerase 3A activity, increases gate opening and closing kinetics in addition to the kinetics of DNA cleavage and religation.  Direct observation of type IA topoisomerase conformational dynamics has contributed to the resolution of mechanistic questions while revealing unanticipated behavior of this important class of topoisomerase.   

Sponsored by the Department of Chemistry & Biochemistry and Co-sponsored with the Biomolecular Sciences Institute and Biochemistry Ph.D. Program