The mechanism of type IA topoisomerases - PubMed (original) (raw)

The mechanism of type IA topoisomerases

N H Dekker et al. Proc Natl Acad Sci U S A. 2002.

Abstract

The topology of cellular DNA is carefully controlled by enzymes called topoisomerases. By using single-molecule techniques, we monitored the activity of two type IA topoisomerases in real time under conditions in which single relaxation events were detected. The strict one-at-a-time removal of supercoils we observed establishes that these enzymes use an enzyme-bridged strand-passage mechanism that is well suited to their physiological roles and demonstrates a mechanistic unity with type II topoisomerases.

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Figures

Fig 1.

Fig 1.

Proposed mechanisms for type I topoisomerases. Shown is a segment of DNA in a (−) supercoiled molecule that is acted on by a type I topoisomerase (gray). (Upper) An enzyme-bridged mechanism for type IA topoisomerases. The free energy of (−) supercoiling promotes the melting of the DNA upon enzyme binding and the stabilization of a (−) crossing of single strands of DNA. After cleavage of one strand of DNA, the enzyme bridges the break by covalent attachment to one end and noncovalent binding to the other. Passage of the intact strand through the break inverts the sign of the crossing. The result is a Δ_Lk_ of 1 per round of catalysis. (Lower) A strand-rotation mechanism, as proposed for type IB topoisomerases. After binding to duplex DNA, the enzyme nicks one strand by addition across the phosphodiester bond and thereby becomes covalently attached to one end of the nick. The other end of the nick is not bound, resulting in rotation of the free end about the intact strand before rejoining of the DNA ends. The result is the relaxation of several supercoils and a nonunity Δ_Lk_ per round of catalysis.

Fig 2.

Fig 2.

The experimental configuration. (A) DNA molecules were tagged at their ends with biotin and digoxygenin, allowing attachment to a streptavidin-coated magnetic bead and an antidigoxygenin-coated glass surface. Translation and rotation of a pair of magnets above the DNA molecule changed the extension of the DNA, which was recorded by tracking of the bead's position with an inverted microscope. (B) The behavior of an 11.5-kb duplex DNA molecule under superhelical stress at two applied forces. At F = 0.26 pN, (+) and (−) superhelical turns equivalently reduced DNA extension through the formation of plectonemic supercoils. Increase of the force to 1.9 pN resulted in an asymmetry of the supercoiling vs. extension curve because denatured regions form at the expense of plectonemes in (−) but not (+) supercoiled DNA. For this DNA, 50 turns caused a |σ| of 0.045. (C) Three different DNAs were used in this study: fully duplex 11.5-kb DNA, 8.7-kb duplex DNA with a symmetric mismatch of 12 nucleotides, and 8.7-kb DNA with an asymmetric bulge of 25 unpaired nucleotides.

Fig 3.

Fig 3.

Relaxation of (−) supercoiled DNA by E. coli topo I. The substrate was fully duplex 11.5-kb DNA. Arrows (↑) indicate the introduction of supercoiling by rotation of the magnets. (A) Topo I relaxes (−) supercoils, as shown by the increase in DNA extension, but cannot relax (+) supercoils. (B) The effect of force on (−) supercoil relaxation. DNA supercoiling density equaled −0.03. The topo I concentration was 380 pM, which was above the K_d of ≈200 pM that we estimated with single-stranded DNA. Topo I is inactive at F = 0.26 pN (gray) but active at F = 0.53 pN (green), where more of the (−) superhelical stress is partitioned into DNA untwisting. To determine topo I activity at F > 1 pN, where DNA extension is independent of (−) superhelical density (Fig. 2_B), we periodically monitored DNA extension at F = 0.26 pN. For example, to measure topo I activity at 2.8 pN (red), we measured the degree of (−) supercoiling of the DNA at 0.26 pN, increased the force to 2.8 pN on a time scale that was fast compared with that of topo I activity, added topo I, and reduced the force to 0.26 pN to remeasure the degree of supercoiling. Thus, we found that topo I remained active at 2.8 pN, but at a reduced rate. At F = 5.4 pN (blue), no topo I activity was detected.

Fig 4.

Fig 4.

Rate of (+) supercoil removal by topo I as a function of the applied force. (A) Variation of the enzymatic rate with force on a DNA construct containing a symmetric mismatch of 12 nucleotides. The data shown are for E. coli topo I (similar results were obtained with T. maritima topo I) and were fit by using v ∼ exp(−_F_δ/k_B_T), yielding δ (the distance DNA moves during the force-sensitive step) of about 10 nm. (B) Variation of the enzymatic rate with force on a DNA construct containing an asymmetric bulge of 25 nucleotides. The data shown are for T. maritima topo I, but similar results were obtained with the E. coli enzyme. The results were fit to a straight line that deviates from a flat line by less than 10%.

Fig 5.

Fig 5.

Step-wise relaxation of (+) supercoiled DNA. The DNA contained a symmetric mismatch of 12 nucleotides. (A and B) Single relaxation events observed in supercoil removal by (A) E. coli topo I (F = 2.0 pN) and (B) T. maritima topo I (F = 1.5 pN) in 0.25 mM MgCl2. The blue points are the raw data, and the red lines are the best-fitted steps. (C) Probability distribution of E. coli topo I step-sizes (n = 610) in seven experiments with forces ranging between 1.0 pN and 2.0 pN. The data were fit to a Gaussian to yield an average value of 〈|Δ_Lk_|〉 = 1.03 ± 0.01. (Inset) Results from a single experiment at 2.0 pN (n = 90); peak fitted to 〈|Δ_Lk_|〉 = 0.97 ± 0.03.

Fig 6.

Fig 6.

Kinetic analysis of E. coli topo I at a force of 2.0 pN. (A) Histogram of times between steps in topo I activity, obtained by fitting the data in the Fig. 5_C_ Inset over a time window _t_av, where t_av was allowed to vary between acquisitions. The exponential fit yields an enzymatic turnover time, τ, of 15.4 ± 3 s, corresponding to a mean rate of DNA elongation, 〈_v_〉, of 2.27 nm/s. From this fit, we estimated that topo I performed a total of 15 ± 3 steps in a period faster than our time resolution t_av. This result agrees well with the 17 fast steps-of-one contained in events contributing 〈|Δ_Lk|〉 ≥ 2 (Fig. 5C Inset). To count these fast steps, a cutoff of 〈|Δ_Lk|〉 = 1.5 was applied to the Gaussian distribution of the data. (B) Examples of the spectral distributions of the fluctuations of DNA extension obtained with (red) and without (gray) topo I. The difference between these two curves was fit to the function A/_f_2 (blue curve), where f is the frequency and A is the amplitude. A is related to the enzymatic rate v and the step-size S (in nm) by A = Sv/2π2. From an average of over eight spectral distributions, we determined a value of 〈_A_〉 = 4.30 nm2 Hz. By using the mean velocity 〈_v_〉 = 2.27 nm/s, we deduced a step-size 〈Δ_Lk_〉 = 1.07 ± 0.23.

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