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Comparative Study
. 2012 Oct 2;109(40):16125-30.
doi: 10.1073/pnas.1206480109. Epub 2012 Sep 18.

A kinetic clutch governs religation by type IB topoisomerases and determines camptothecin sensitivity

Yeonee Seol  1 , Hongliang Zhang, Yves Pommier, Keir C Neuman
Affiliations
Comparative Study

A kinetic clutch governs religation by type IB topoisomerases and determines camptothecin sensitivity

Yeonee Seol et al. Proc Natl Acad Sci U S A. .

Abstract

Type IB topoisomerases (Top1Bs) relax excessive DNA supercoiling associated with replication and transcription by catalyzing a transient nick in one strand to permit controlled rotation of the DNA about the intact strand. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Top1B (nTop1). Previous work on vaccinia Top1B led to an elegant model that describes a straightforward dependence of rotation and religation on the torque caused by supercoiling. Here, we used a single-molecule DNA supercoil relaxation assay to measure the torque dependence of nTop1 and its inhibition by CPT. For comparison, we also examined mitochondrial Top1B and an N-terminal deletion mutant of nTop1. Despite substantial sequence homology in their core domains, nTop1 and mitochondrial Top1B exhibit dramatic differences in sensitivity to torque and CPT, with the N-terminal deletion mutant of nTop1 showing intermediate characteristics. In particular, nTop1 displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torque-independent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation and the efficiency of CPT binding. This "kinetic clutch" mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cartoon of experimental setup (not to scale) and an example trace. (A) A DNA molecule is attached between the cover glass of a flow cell and a magnetic bead (green). Force is controlled by moving the magnet assembly above the flow cell. Rotating the magnetic bead by turning the magnets twists the DNA and generates supercoils. (B) DNA extension as a function of time and step-finding fit. DNA extension traces were fitted with a custom written step-finding routine based on a t test algorithm (40). The fitting routine extracts the extension change, duration, and linear velocity for each relaxation event and the duration of pauses between events. These phases of motion correspond to cleavage (green arrow), relaxation, and religation (blue arrow) depicted in the cartoon of Top1B enzyme bound to DNA.
Fig. 2.
Fig. 2.
Torque dependence of the supercoil relaxation rate. The energy barriers, EB, and transition angles, θ, were obtained from the fits of the torque-dependent relaxation rate (ke) to the barrier-crossing model (10, 11): formula image, where r0 is the rate of thermal fluctuations, r0 ∼ (β ηl3)−1 [ where η is the buffer viscosity (∼10−3 Pa⋅s) and l is the radius of DNA (1 nm)], EB is the energy barrier, Γ is the external torque, θ is the transition angle, and β = 1/kBT is the inverse of the thermal energy (SI Materials and Methods). The fitted values were as follows: EB (pN⋅nm): 84.7 ± 0.6 for Top1mt, 83.8 ± 0.6 for nTop1, and 84.1 ± 0.2 for Top68; θ (rad): 0.39 ± 0.02 for Top1mt, 0.27 ± 0.03 for nTop1, and 0.31 ± 0.02 for Top68. All reported errors are SDs.
Fig. 3.
Fig. 3.
Torque-dependent relaxation step size and religation probability. (A) Relaxation step sizes of Top1mt, nTop1, and Top68 are plotted as a function of torque and fitted with Eq. 1 (solid lines, left axis). The religation probabilities corresponding to the inverse of the relaxation step sizes are shown with fits (dashed lines, right axis). The relaxation step sizes were obtained by fitting the relaxation step size distribution at each torque with a single exponential (Fig. S5). (Inset) Relaxation step sizes of Top1mt, nTop1, and Top68 at positive (9.3 pN⋅nm) and negative (−9.3 pN⋅nm) torques. (B) Ratio kf/kL and kR for three Top1Bs obtained from the fits of the relaxation step sizes as a function of torque to Eq. 1: formula image : 56 ± 6 for Top1mt, 39 ± 2 for nTop1, and 35 ± 4 for Top68; formula image (s−1): 10 ± 3 for Top1mt, 4 ± 2 for nTop1, and 6 ± 3 for Top68. All reported errors are SDs.
Fig. 4.
Fig. 4.
Cleavage kinetics. (A) Cleavage rates of the three Top1B enzymes were calculated with Eq. 2 using the mean cleavage time <Tc> averaged over all torques (Fig. S8) and the previously obtained parameters (ke, kR, and kf/kL). The calculated cleavage rates, kc (s−1), were as follows: 0.35 ± 0.04 for Top1mt, 0.83 ± 0.04 for nTop1, and 0.85 ± 0.03 for Top68. All reported errors are SEs. (B) Ratio of the ligation-to-cleavage probabilities at zero torque as a function of kf. Top1mt shows a twofold higher pL/pc at kf ≥ 300 s−1 in comparison to nTop1. Because rotation is the rate-limiting step in the relaxation process, kf is larger than ke for each enzyme. The shaded region corresponds to kf < 300 s−1, the minimal kf consistent with the slowest relaxation rate.
Fig. 5.
Fig. 5.
CPT sensitivity of human Top1B enzymes relaxing positively supercoiled DNA. (A) Normalized relaxation rate distributions of three Top1B enzymes in the presence and absence of 5 μM CPT and 100 μM CPT for Top1mt. Relaxation rates of nTop1 and Top68 decreased at 5 μM CPT, whereas a comparable decrease in the relaxation rate of Top1mt required 100 μM CPT. (B) Top1B-DNA cleavage complex formation. With nTop1 (Right), cleaved DNA was visible at the lowest CPT concentration (0.1 μM), whereas with Top1mt (Left), cleaved DNA was minimally visible up to 10 μM CPT and became comparable to that observed with nTop1 at 100 μM CPT. C* indicates a control reaction that contains only DNA. (C) Cleavage complex formation as a function of CPT concentration. DNA cleavage was quantified by the intensity in the cleavage band normalized by total DNA (uncleaved and cleaved DNA) corrected by the intensity of the control band.
Fig. 6.
Fig. 6.
Proposed kinetic clutch model for Top1B. In this scheme, Top1B (T) reversibly binds DNA (D), forming a binary complex (T⋅D) in which the DNA is cleaved at the rate kc. The cleaved complex is initially in a religation-competent state (T⋅Dc) separated from the rotation-competent state (T⋅Dc*) by a torque-independent energy barrier, corresponding to a protein conformational change or a radial DNA distortion (orange and red states). The Top1B–DNA complex fluctuates between the religation-competent (T⋅Dc) and rotation-competent (T⋅Dc*) states at the torque-independent rates kR and kf. From the religation-competent state T⋅Dc, the DNA can be religated at rate kL. DNA rotation corresponds to escape from the T⋅Dc* state at the torque-dependent rate ke. Each rotation of the DNA corresponds to one cycle of T⋅Dc to T⋅Dc* and back to T⋅Dc as denoted by the blue-shaded box. The corresponding reaction coordinates are schematically represented below. The free end of the DNA rotates from the state T⋅Dc* (red dot) but is prevented from further rotation once it enters the state T⋅Dc (orange dot). A reversible radial motion (i.e., crossing an energy barrier orthogonal to rotation) is required to return the complex to the rotation-competent state. Although the model is based on positive supercoil relaxation, we speculate that a similar mechanism holds for negative supercoil relaxation. Based on the “intercalated model,” CPT likely binds Top1B in the religation-competent state (T⋅Dc) in which the two DNA ends are aligned.
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