Mechanochemical analysis of DNA gyrase using rotor bead tracking

Gore, Jeff; Bryant, Zev; Stone, Michael D.; Nöllmann, Marcelo; Cozzarelli, Nicholas R.; Bustamante, Carlos

doi:10.1038/nature04319

Cited by 177 publications

(165 citation statements)

References 29 publications

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“…(Fig. 1b) have been used to study the mechanical properties of nucleic acids (Strick et al 1996;Abels et al 2005) and the mechanisms of enzymatic reactions with DNAs as substrates (Crisona et al 2000;Revyakin et al 2003;Dessinges et al 2004;Gore et al 2006). In a recent study, Abels et al (2005) ligated a long dsRNA (4·2 kb or 8·3 kb) with two 0·4 kb pieces of dsRNA, each of which was labeled by multiple digoxigenins or biotins.…”

Section: Instrumentationmentioning

confidence: 99%

Determination of thermodynamics and kinetics of RNA reactions by force

Tinoco

Bustamante

2006

Quart. Rev. Biophys.

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107

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Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.

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Section: Instrumentationmentioning

confidence: 99%

Determination of thermodynamics and kinetics of RNA reactions by force

Tinoco

Bustamante

2006

Quart. Rev. Biophys.

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107

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“…The development reported here expands on the current arsenal of hybrid single molecule techniques combining force and other observables (8,(25)(26)(27). Unlike DNA or RNA hairpins, where forces on the order of 15 pN are necessary to induce mechanical unzipping (10,11), the conformations of HJs could be biased at 0.5 pN or lower.…”

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confidence: 99%

Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

Hohng

Zhou

Nahas

et al. 2007

Science

269

299

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Despite the recent advances in single molecule manipulation techniques, purely mechanical approaches can not detect subtle conformational changes in the biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence which allowed us to examine the effect of sub-pN forces on the nanometer scale motion of the Holliday junction (HJ) at 100 Hz bandwidth. The HJ is an exquisitely sensitive force sensor whose force response is amplified with an increase in its arm lengths, demonstrating a lever-arm effect at the nanometer length scale. Mechanical interrogation of the HJ in three different directions helped elucidate the structures of the transient species populated during its conformational changes. This method of mapping two dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes.Many biological processes are dependent on tension. In recent years, single molecule force measurements have shown directly that biochemical reactions can be influenced by applied force (1). Yet, purely mechanical tools can not detect small scale conformational changes unless strong enough and persistent force is applied. At weak forces, the flexible tether connecting the mechanical probe to the biological molecule is not stretched enough to transmit small movements. This is unfortunate because weak and transient forces are likely more prevalent in vivo but the experimental limitations confine single molecule mechanical studies to examining the effect of relatively large forces. We aimed to study the effect of weak external forces on the biomolecular conformational dynamics by combining single molecule fluorescence resonance energy transfer (smFRET) (2-4) with manipulation using optical trap (5). smFRET has high spatial resolution (≤ 5 Å) (6, 7)) and can be measured at arbitrarily low forces. Previous attempts to combine FRET and optical trap using the DNA hairpin as a model system (8, 9) did not reveal new information because the hairpin unzips at high forces (~ 15 pico Newton (pN)), a regime that had been extensively investigated using force-based techniques (10, 11). Here, we report an approach to detect nanometer-* To whom correspondence should be addressed. (tjha@uiuc.edu). † Present Address: Department of Physics and Astronomy, Seoul National University, Seoul 151-742, Korea. scale motion at sub-pN forces. We used the approach to gain insight into the reaction landscape of the Holliday junction (HJ) by gently stretching it along different directions. NIH Public AccessThe HJ is a four-stranded DNA structure that forms as an intermediate during recombination (12). To understand the mechanisms of cellular enzymes that function with the HJ, a detailed description of the static and dynamic structural properties of the HJ itself is needed. In the absence of added ions the HJ adopts an open structure, where the four helical arms point toward the corners of a square (13, 14) (Fig. 1A). In the presence ...

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“…Motivated by recent single-molecule studies that have revealed the operation of various topoisomerases in real time (19)(20)(21)(22)(23), we have observed the activity of purified reverse gyrase from S. tokodaii (4) under an optical microscope at 71°C (Fig. 1).…”

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confidence: 99%

Direct observation of DNA overwinding by reverse gyrase

Ogawa

Yogo

Furuike

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70°C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71°C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50°C. The unloaded reaction rate at 71°C exceeds five turns per second, which is >10 2 -fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s −1 , indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼5 pN·nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding.reverse gyrase | topoisomerase | magnetic tweezers | DNA overwinding | torsion R everse gyrase, discovered in 1984 in a hyperthermophilic archaeon Sulfolobus (1) which was later classified as Sulfolobus tokodaii (2), is a unique DNA topoisomerase that can introduce positive supercoils into DNA (3-7). The only other enzyme that has the gyration activity is DNA gyrase, which introduces negative supercoils. Although DNA gyrase belongs to type II topoisomerase, which changes the linking number (Lk) of dsDNA by two by cutting and religating both strands simultaneously, reverse gyrase is of type IA topoisomerase (topo IA), where one strand is cut to allow the passage of the other, resulting in the Lk changes in steps of one (3-7). Reverse gyrase is found in hyperthermophilic archaea and eubacteria, and the positive supercoiling activity requires a temperature above 70°C (8, 9). The enzyme is a 130-kDa single polypeptide, a fusion complex of two domains (10): The carboxyl terminal half is related to topo IA, whereas the amino terminal half has an ATP-binding site and is akin to helicase, although neither the whole enzyme nor the isolated helicase-like domain shows genuine helicase activity (11). A crystal structure of the full-length reverse gyrase (12) and more recent structures with additional features (13) all support the basic two-domain construct. The physiological roles of reverse gyrase are not yet fully clear, although positive supercoiling is expected to protect DNA from denaturation at the growth temperatures of hyperthermophiles.Reactions of reverse gyra...

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Mechanochemical analysis of DNA gyrase using rotor bead tracking

Cited by 177 publications

References 29 publications

Determination of thermodynamics and kinetics of RNA reactions by force

Determination of thermodynamics and kinetics of RNA reactions by force

Fluorescence-Force Spectroscopy Maps Two-Dimensional Reaction Landscape of the Holliday Junction

Direct observation of DNA overwinding by reverse gyrase

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