Binding Kinetics Oppositely Regulates type II Topoisomerase Relaxation and Decatenation Activities

TL;DR

This study addresses how type II topoisomerase (Topo II) modulates genome topology through decatenation and relaxation, investigating whether binding kinetics govern these distinct catalytic outputs. The authors combine single-molecule measurements of DNA binding kinetics, bulk decatenation/relaxation assays, and coarse-grained molecular dynamics to test how monovalent cations alter $k_{ extrm{off}}$ and, consequently, topo II activity. They find that higher salt accelerates decatenation while slowing relaxation, a divergence explained by simulations showing a trade-off between efficient 3D target search and processive, long-lived DNA engagement. The results reveal binding kinetics as a regulatory layer that can differentially tune topo II activities in vivo, with implications for PTMs and protein interactions that localize and modulate topo II in cellular contexts.

Abstract

Type II Topoisomerases (topo II) are critical to simplify genome topology during transcription and replication. They identify topological problems and resolve them by passing a double-stranded DNA segment through a transient break in another segment. The precise mechanisms underpinning topo IIs ability to maintain a topologically simple genome are not fully understood. Here, we investigate how binding kinetics affects the resolution of two distinct forms of topological entanglement: decatenation and torsional relaxation. First, by single-molecule measurements, we quantify how monovalent cation concentration affects the dissociation rate of topo II from DNA. Second, we discover that increasing dissociation rates accelerate decatenation while slowing down relaxation catalytic activities. Finally, by using molecular dynamics simulations, we uncover that this opposite behaviour is due to a trade-off between search of target through facilitated diffusion and processivity of the enzyme in catenated versus supercoiled DNA. Thus, our findings reveal that a modulation of topo II binding kinetics can oppositely regulate its topological simplification activity, and in turn can have a significant impact in vivo.

Figures (4)

• Figure 1: a. Topo II search on DNA, including 1D and 3D diffusion and intersegmental jumps. On and off rates, and 1D diffusion constant are expected to be salt dependent. b. Topo II present exposed positive surface charges that stabilise the binding with negatively charged DNA substrate. Its intrinsically disordered CTD also display positive charges that can bind to dsDNA (see SI). red = negative, blue = positive charges. c. Electrophoretic mobility shift assay (EMSA) performed on a 51 bp dsDNA varying topo II concentration from 0 to 140nM, at 125 mM NaCl. d. Quantification of the bound dsDNA fraction in the EMSA, returning an approximate $k_D \simeq 50$ nM.
• Figure 2: Binding kinetics of topo II is modulated by monovalent cations.a Diagram of force-stretched DNA. DNA tethered between trapped beads, topo II complex is labeled using atto647. c-f Kymographs of atto647-labelled topo II binding, diffusing and unbinding from a stretched $\lambda$DNA using optical tweezers at 75-150 mM [NaGlu]. g Quantification of inverse dwell time, or $k_{\textrm{off}}$ , as a function of monovalent cation concentration. Each concentration includes analysis of more than 100 single-molecule tracks. All pair-wise distributions are significantly different (p value $< 0.01$).
• Figure 3: Topoisomerase decatenation and relaxation activities are oppositely regulated by salt concentration.a. To quantify decatenation activity we monitor the disassembly of kDNA as a function of various [NaCl]. b. Gel electrophoresis of 20 ng/ul kDNA after 30 minutes incubation with 3.45 ng/ul of topo II. The first lane shows the control sample without protein, and the following five lanes correspond to reactions performed in buffers containing [NaCl] = 25, 48, 87, 120, and 148 mM, respectively. c. Quantification of decatenated minicircles as a function of [NaCl]. d. To quantify the relaxation activity we monitor the topology of a negatively supercoiled pBR322 plasmid as a function of various [NaCl]. e. Chloroquine gel electrophoresis of 20 ng/ul pBR322 after 30 minutes incubation with 3.45 ng/ul of topo II. As in panel b, the first lane corresponds to the control sample without protein, and the remaining lanes show reactions carried out at [NaCl] = 25, 48, 87, 120, and 148 mM. Notably the amounts of supercoiled and relaxed DNA at 120mM and 87mM NaCl are comparable, nevertheless the supercoil distributions are visibly different, showing a slowed relaxation effect at higher NaCl concentrations. f. Quantification of relaxed topoisomer fraction as a function of [NaCl].
• Figure 4: Molecular Dynamics simulations with salt-dependent $k_{\textrm{off}}$ capture the opposite topological regulation.a. Snapshots from MD simulations of a model kDNA from Ref. He2023 before and after decatenation reaction. b. Fraction of catenated minicircles as a function of time and for different values of off-rate. Shaded area represents the standard deviation over 50 independent replicas. c. Fraction of catenated minicircles at fixed time ($10^4 \tau_B$) and as a function of $k_{\textrm{off}}$ showing enhanced decatenation at larger off-rate. d. Snapshot from MD simulations of a supercoiled DNA chain (model from Ref. Brackley2014supercoilSmrek2021. e. Step-wise change in writhe monitored through the simulation. Snapshots at the top, plot of writhe at the bottom. f. Average writhe $\langle \mathcal{W} \rangle$ as a function of time and for different values of off-rate. The shaded area represents the standard deviation across 50 independent replicas. g. Fraction of supercoiled topoisomers (defined as those with $\mathcal{W}/N > 0.05$) as a function of off-rate (SC = fully supercoiled state).