Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

Abstract

Topoisomerase V is a type I topoisomerase without structural or sequence similarities to other topoisomerases. Although it belongs to the type I subfamily of topoisomerases, it is unrelated to either type IA or IB enzymes. We used real-time single-molecule micromechanical experiments to show that topoisomerase V relaxes DNA via events that release multiple DNA turns, employing a constrained swiveling mechanism similar to that for type IB enzymes. Relaxation is powered by the torque in the supercoiled DNA and is constrained by friction between the protein and the DNA. Although all type IB enzymes share a common structure and mechanism and type IA and type II enzymes show marked structural and functional similarities, topoisomerase V represents a different type of topoisomerase that relaxes DNA in a similar overall manner as type IB molecules but by using a completely different structural and mechanistic framework.

Fig. 1.

Relaxation mechanisms and single DNA molecule calibration. (A) Possible mechanisms of DNA relaxation by topo V. Two possible mechanisms for DNA relaxation have been proposed for type I topoisomerases. Upon DNA binding (a), the relaxation cycle by topo V may proceed either by a strand passage mechanism (b) or a swiveling mechanism (c). The expected step-size distribution is shown for each case. The coordinates of topo-61 (PDB ID code 2CSB) were used to model possible interactions of topo V with DNA (5). (B) DNA calibration. The extension of a 9.7-kb DNA was monitored at different stretching forces. At a low force of 0.2 pN (red circles), the extension of DNA decreases irrespective of the sign of supercoiling because of the formation of plectonemic supercoils. At 0.5 pN (green triangles), DNA extension still decreases irrespective of the direction of rotation of the magnet, although plectonemes coexist with denatured DNA for negative supercoils. At high forces (1 pN, blue squares; 2.5 pN, black diamonds), DNA extension decreases only with the introduction of positive supercoils. The DNA denatures for negative supercoils, and there is no change in DNA extension with introduction of negative rotations. Events only were considered for analysis only when they fell in the linear range of the DNA calibration curve.

Fig. 2.

Monitoring of real-time DNA relaxation events and step-size distributions. (A) Real-time relaxation events on positively supercoiled DNA are shown at 1.5 pN in the presence of glycerol. An increase in DNA extension upon addition of topo-78 was observed. Each step corresponds to a relaxation event representing one cleavage/religation cycle. The change in linking number is of different size for different events. Hence, topo V relaxes DNA in steps of n supercoils. The dashed lines mark the extension limits used to select events for further analysis in this particular experiment. (B) A step-size distribution of topo-78 acting on a single DNA molecule is shown for events monitored at a force of 0.5 pN in presence of glycerol when 45 plectonemic supercoils were incorporated. A similar shape distribution was observed at all forces studied. The mean step size for each distribution was computed numerically by using the method of Koster et al. (14), yielding a mean ΔLk of 12.3 ± 1.8. (Inset) Step-size distribution for topo-78 measured in absence of glycerol when 45 plectonemic supercoils were applied at a force of 0.5 pN. The line was fit assuming an exponential decay. Error bars represent the square root of the number of events.

Fig. 3.

Dependence of mean step size on force. The mean step size (〈ΔLk〉) was plotted as a function of applied force and shows an increase in the mean step size with increasing force. The dashed line serves as a visual guide only. The error bars represent the standard error [standard deviation/(number of events)^1/2].

Fig. 4.

Linear and angular velocity dependencies on force and torque. (A) Linear velocity (μm/sec) has a nonlinear dependence on force, indicating that bead translational drag forces do not limit the rate of relaxation. The dashed line serves as a visual guide and does not correspond to a fit. (Inset) Plot of the angular relaxation velocity versus force whose dependence is linear over the range of forces studied. The linear regression fit has a correlation coefficient of 0.991. (B) Angular relaxation velocity ω (rad/sec) initially increases linearly with torque, indicating that rotational friction is the limiting source of friction in the relaxation over the range of forces studied (SI). The line corresponds to a fit of the data to the expression relating angular velocity to torque (SI), with positive and negative angular barrier widths set equal (θ₊ = θ₋), k_BT = 4.1 pN·nm, and ηℓ³ = 10⁻⁹ pN·nm·sec. From the fit (reduced χ² value = 1.4), the energy barrier (E_B) is equal to 80.1 ± 0.7 pN·nm or 11.5 ± 0.1 kcal/mol. (C) The mean step size increases with increasing angular relaxation velocity. The line corresponds to a fit of the data to an equation describing the probability per turn that religation does not occur (P₁), noting that 〈ΔLk〉 = −1/(1 − P₁) (see SI). Using this model, we obtain values of kδ = 361.4 ± 132.4 sec⁻¹ and k′δ = 33.8 ± 6.7 sec⁻¹, respectively, where k and k′ are the cleavage and religation rates and δ is the angular range where cleavage and religation occur. The fit indicates that 〈ΔLk〉 = 11.7 ± 1.0 at zero angular velocity. In all cases, the error bars represent the standard error.