Two gates versus three gates: Mechanistic differences in DNA relaxation and decatenation by topoisomerases II and VI

• Applicant: Professorin Dr. Dagmar Klostermeier
• Subject Area: Biochemistry
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 571438647

Topoisomerases are enzymes that inter-convert different DNA topoisomers. They catalyze the introduction and removal of supercoils, catenation and decatenation, and knotting and unknotting of DNA. Inter-conversion of different topoisomers requires cleavage of one or both strands of the DNA. Type II topoisomerases cleave both strands of their DNA substrate, and employ a strand-passage mechanism, in which both strands of a DNA duplex (G-segment) bound to the enzyme are cleaved, and the coordinated closing and opening of transient protein-protein interfaces (gates) then captures a second duplex (T-segment) and drives its passage through the gap in the cleaved G-segment. The type IIA topoisomerase Topo II and the type IIB enzyme Topo VI both catalyze the ATP-dependent relaxation and decatenation of DNA. However, they differ structurally: While Topo II is a homodimer of two identical TOP2 subunits, Topo VI is a heterotetramer formed by two Top6A and two Top6B subunits. The Topo II dimer forms three gates, termed N-gate, DNA-gate and C-gate. In contrast, the Topo VI heterotetramer forms only two gates, an N-gate and a DNA-gate, but lacks a C-gate. The structural differences strongly suggest differences in the mechanisms of the two enzymes. These differences, as well as possible differences between the relaxation and decatenation reactions, are currently unknown. In our work on the bacterial type II topoisomerases gyrase and Topo IV, and in proof-of-principle experiments with Topo II and Topo VI, we established an extensive toolbox to study the mechanism of DNA supercoiling, relaxation, and decatenation by these enzymes. Building on these resources, we will interrogate the mechanistic differences of ATP-dependent relaxation and decatenation of DNA by Topo II and Topo VI. We will dissect the conformational changes in Topo II and Topo VI in single-molecule FRET experiments, both in ATP-dependent relaxation and in DNA decatenation. Specifically, we will define conformational changes induced in Topo II and Topo VI by ATP and DNA binding, with a focus on gate opening and closing. We will investigate the effects of each ATP binding and hydrolysis event, of different DNAs, and of both DNA cleavage events on enzyme conformation. We will determine the stabilities of the gates, their propensities to open, and the events triggering opening and closing. We will then use single-molecule FRET conformational changes beyond gate opening and closing. Finally, we will address the coordination of the conformational changes occurring. Collectively, the research proposed here will define the mechanistic differences (1) between ATP-dependent DNA relaxation by Topo II and Topo VI, (2) between the decatenation reactions catalyzed by the two enzymes, as well as (3) the differences between ATP-dependent relaxation and decatenation for each enzyme. Thereby, we will uncover common features and differences in the three-gate- and two-gate mechanisms employed by Topo II and Topo VI.

• DFG Programme: Research Grants