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330 Force spectroscopy of single DNA and RNA molecules Mark C Williams* and Ioulia Rouzina† Experiments in which single molecules of RNA and DNA are stretched, and the resulting force as a function of extension is measured have yielded new information about the physical, chemical and biological properties of these important molecules. The behavior of both single-stranded and double-stranded nucleic acids under changing solution conditions, such as ionic strength, pH and temperature, has been studied in detail. There has also been progress in using these techniques to study both the kinetics and equilibrium thermodynamics of DNA–protein interactions. These studies generate unique insights into the functions of these proteins in the cell. Addresses *Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, 111 Dana Research Center, Boston, MA 02115, USA; e-mail: [email protected] † Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St Paul, MN 55108, USA Current Opinion in Structural Biology 2002, 12:330–336 0959-440X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations AFM atomic force microscopy bp base pairs ds double-stranded NC HIV-1 nucleocapsid protein ss single-stranded Introduction In the past decade, several techniques have been developed for measuring small forces acting on single DNA and RNA molecules. In these experiments, one end of the molecule is held at a fixed position, while the other end of the molecule is extended at constant force or to a fixed position. These techniques include atomic force microscopy (AFM) [1], optical tweezers [2] and magnetic tweezers [3,4•], as shown in Figure 1. All of these instruments are able to measure the force required to stretch DNA under various conditions. In optical tweezers, one end of a single DNA or RNA molecule is attached to a polystyrene bead in an optical trap, while the other end is held a fixed distance from the trap (Figure 1a). The resulting force on the bead in the trap is measured over a force range of 0.1–150 pN. In magnetic tweezers, one end of a single DNA molecule is attached to a magnetic bead, while the other end is attached to a glass surface (Figure 1b). The magnetic field exerts a constant force, so the resulting extension of the molecule as a function of force can be measured. This method can be used to measure forces well below 1 pN. In AFM experiments, DNA molecules are attached to a fixed surface at one end and a cantilever at the other end (Figure 1c). As the fixed surface is pulled away from the cantilever, the deflection of the cantilever can be used to determine the force required to stretch the DNA molecule. The resolution of this method is about 5 pN, but it can also be used to measure forces in the nanonewton range. This wide range of techniques has been used to study several regimes of DNA stretching behavior in detail and has also been extended to study DNA–protein and DNA–drug interactions. In addition, high-resolution force measurements have been obtained from the stretching of single RNA molecules, suggesting that these methods can be extended to the study of sequence-specific RNA–protein interactions. Stretching nucleic acids at low forces: DNA elasticity, and DNA and RNA unzipping The force/extension curve of double-stranded (ds) DNA can be fit to the following equation: FPds bds 1 1 = • − 1 + max max kBT 4 (1 − bds /bds + F/K ds ) bds (1) where Pds is the persistence length, Kds is the elastic stretch modulus, bds is the extension of the molecule per base pair and is the dsDNA contour length per base pair. F is the force required to extend the molecule, kB is Boltzmann’s constant and T is the temperature. This expression is an interpolation between exact solutions of the worm-like chain model at high and low forces. It describes the persistence length and stretch modulus of dsDNA to within 5% at all extensions. A more accurate interpolation has been proposed by Bouchiat et al. [5], which can be used when very accurate force/extension data are available in the entire range of forces. In 1997, Baumann et al. [6] used the high-force limit of Equation 1 to describe the persistence length and stretch modulus of dsDNA as a function of monovalent salt concentration. The observed increase in persistence length with decreasing monovalent salt concentration was in agreement with the predictions of polyelectrolyte theory. In contrast to expectations from a theoretical model of DNA as a classical homogeneous rod, the authors also showed that the stretch modulus of dsDNA decreased with decreasing salt concentration. As the stiffness of DNA has a contribution from the repulsion between the negatively charged phosphate groups that make up its backbone, Podgornik et al. [7•] developed a theory to describe the expected salt dependence of the stretch modulus and persistence length. However, their predictions for the stretch modulus of dsDNA disagreed with the measurements of Baumann et al. [6]. Recent measurements of the salt dependence of DNA stretching, coupled with a new fitting procedure using the theory of Podgornik et al. [7•] to relate Pds and Kds, however, indicate that the stretch modulus data are consistent with the theory of Podgornik et al. [7•] (JR Wenner, MC Williams, I Rouzina, VA Bloomfield, unpublished data). Force spectroscopy of single DNA and RNA molecules Williams and Rouzina 331 Figure 1 Single-molecule force spectroscopy. (a) In an optical tweezers instrument, one or two laser beams are focused to a small spot, creating an optical trap that attracts polystyrene beads. Single DNA molecules are attached at one end to a bead in the trap, while the other end is attached to a moveable surface, which, in this example, is another bead on a glass micropipette. As the DNA molecule is stretched by moving the micropipette, the resulting force on the bead in the trap is measured. (b) In a magnetic tweezers instrument, single DNA molecules are attached at one end to a glass tube, while the other end is attached to a magnetic bead. Magnets located outside the tube generate a magnetic field that exerts a constant force on the magnetic bead. The extension of the DNA molecule as a function of the applied force is then measured. (c) In an AFM experiment, single DNA molecules are attached to a surface. The other end of one of these molecules is attached to a cantilever. As the surface is pulled away from the cantilever, the deflection of the cantilever is monitored by measuring the position of a reflected laser beam, which determines the force required to stretch the attached DNA molecule. In the presence of a critical concentration of multivalent ions, dsDNA condenses and forms compact rods and toroidal structures [8]. Single-molecule stretching methods have been used to measure the forces that cause this DNA condensation. If the two ends of a DNA molecule are stretched, they are prevented from condensing. The force required to prevent condensation, the attractive condensation force, has been shown to be constant, with a magnitude between 1 and 4 pN [9,10]. These results are consistent with the low attractive condensation force measured in bulk experiments. In contrast to these observations, high concentrations of the nonspecific DNAbinding protein integration host factor (IHF) induce compaction by locally bending the DNA, but do not induce collapse [11•]. It is therefore likely that the nonspecific binding of this protein to DNA strongly affects the large-scale structure of the bacterial nucleoid. (a) Laser beam Microscope objectives Polystyrene bead Laser beam DNA molecule Glass micropipette (b) S N → F N S Glass surface Bead DNA (c) Detector Laser Precise measurements of the elasticity of single-stranded (ss) DNA have been made at very low forces. The results show a strong sequence dependence, indicating the importance of secondary structure formation at low forces [4•,12]. Bockelmann et al. [13] have demonstrated that the force required to cooperatively remove hairpin secondary structure as DNA is unzipped varies between 12 and 15 pN, depending on the sequence of the DNA. This is consistent with the observed sequence dependence of ssDNA elasticity at forces less than 15 pN. Several theoretical treatments of DNA unzipping have recently been published [14–16]. Liphardt et al. [17••] have recently demonstrated the force-induced unfolding/refolding of small RNA hairpins (Figure 2). In these experiments, they unzipped various single-molecule hairpins by pulling on their ends with DNA–RNA hybrid handles attached to beads. They measured forces of about 15 pN when pulling apart simple double-stranded portions of RNA, similar to earlier measurements of the forces needed for DNA unzipping. Cantilever DNA Piezoelectric stage Current Opinion in Structural Biology Pulling and relaxing force curves for some types of hairpin were indistinguishable at low pulling rates, thus indicating thermodynamic reversibility. The area under the reversible force/extension curves yields a direct measurement of the equilibrium free energy of structure formation. This area for a simple 49 bp hairpin structure 332 Nucleic acids Figure 2 (a) ∗ 20 +Mg –Mg 20 ∗ 15 15 15 10 ∗ 10 200 ∗ ∗ 10 Force (pN) Force (pN) (b) 20 250 5 5 100 nm 100 150 (c) 200 Extension (nm) 2 nm 5′ 250 13 nm 3′ 5′ P5a A-rich bulge P5c 3′ A-rich bulge P5c Three-helix junction P5b P5b 26 nm 3′ Force/extension curves for the P5abc domain of the T. thermophila ribozyme. (a) In the presence of Mg2+, an RNA structural transition occurs as the RNA molecule is stretched to about 20 pN (blue line) and the relaxation curve (green line) shows hysteresis. The blue arrow indicates the typical unfolding force when stretching — the green arrow shows a refolding transition upon relaxation. Inset: a detailed stretching curve (light blue line) of the P5abc RNA structure reveals the presence of intermediate structural elements stabilized by metal binding (red stars). The lower blue arrow shows the lowest unfolding force. In other stretches (dark blue line), unfolding occurs suddenly at a much higher force (top blue arrow). (b) RNA stretching curves (blue lines) differ significantly in the presence and absence of Mg2+, whereas the relaxation curves (green lines) exhibit similar intermediate transitions at lower forces (black stars and green arrows). These intermediate transitions are also observed when stretching this RNA in the absence of Mg2+. (c) A model for the unfolding of P5abc in the presence of Mg2+, in which two possible unfolding paths are shown. The blue arrow shows an unfolding path in which the molecule suddenly unfolds and increases its length to 26 nm, consistent with data indicated by the blue arrow in (a). A two-step model is shown by the red arrows, in which an intermediate state 13 nm in length is formed during stretching. Transitions to this intermediate state are indicated by green arrows in (a) and (b). (Reproduced with permission from [17••].) was in good agreement with the theoretical predictions of the MFOLD method [18]. Another structure, the P5abc domain of the Tetrahymena ribozyme, forms tertiary contacts in the presence of Mg2+. As shown in Figure 2, the resulting force/extension curves map out the secondary structure of such an RNA molecule and identify metalbinding pockets. This will be an extremely valuable technique for the study of RNA folding, as well as RNA–protein interactions, as will be discussed below. Stretching DNA at high forces: DNA overstretching and strand separation Torsionally relaxed DNA As a single molecule of dsDNA is stretched beyond its B-form contour length, the force required to further stretch the molecule increases dramatically. If one end of the DNA molecule is allowed to rotate freely, a cooperative overstretching transition occurs at about 65 pN, after which very little additional force is required to stretch the Force spectroscopy of single DNA and RNA molecules Williams and Rouzina To test the force-induced melting model, Williams et al. [28] measured DNA overstretching as a function of pH. As extremely high and low pH lower the melting temperature of dsDNA, the overstretching force should also decrease if melting occurs during the transition. This decrease in the overstretching force was demonstrated and the value of the change in entropy of DNA upon melting, determined from the ratio of the change in overstretching force to the change in melting temperature as a function of pH, was in agreement with calorimetric measurements of this parameter at room temperature. As a further test, Williams et al. [29•] also measured the temperature dependence of DNA overstretching. Although their data were consistent with earlier temperature-dependent measurements using AFM [26•], the high-resolution data obtained using optical tweezers allowed them to directly calculate the helix-coil transition free energy as a function of temperature from the force/extension curves. The resulting parameters describing this temperature dependence — the heat capacity of DNA upon melting and the entropy of DNA upon melting at the melting temperature — were in very good agreement with independent calorimetric measurements of these parameters. Finally, measurements of the Figure 3 100 80 Force (pN) molecule to 1.7 times its contour length [2,19]. To describe this transition, a model of overstretched DNA as a new double-stranded form of DNA, referred to as S-DNA, was proposed [19]. Although models describing S-DNA did predict an overstretching transition, the predicted transition was less cooperative and occurred at a higher force than that observed experimentally [20–22]. Rouzina and Bloomfield [23,24] have proposed an alternative model for DNA overstretching as a force-induced melting process. In this model, the base pairs holding the two DNA strands together break as the DNA unwinds during the transition. This model was shown to be consistent with all available data on the dependence of DNA overstretching on changes in solution conditions such as ionic strength and temperature. It has also been shown that poly(dG•dC)poly(dG•dC) has an overstretching transition about 30 pN higher than that of poly(dA•dT)poly(dA•dT) [25]. This result is consistent with the difference in melting temperature between these molecules. However, the authors also observed an additional strand separation transition at forces higher than the overstretching force. In later work [26•], they showed that this strand separation force depended on the rate at which the dsDNA was stretched. In the force-induced melting theory, the overstretching transition is an equilibrium melting transition, whereas the second transition at higher force is a nonequilibrium strand separation transition, during which the last base pairs holding the two strands together are irreversibly broken. A rate-dependent force is expected when single bonds are irreversibly broken [27]. Thus, the rate dependence observed when stretching DNA at forces greater than 65 pN indicates that this portion of the transition is irreversible, consistent with the idea that these forces are due to irreversible strand separation. 333 60 40 20 0 0.2 0.3 0.4 0.5 0.6 DNA extension (nm/bp) Current Opinion in Structural Biology The effect of changing solution conditions and protein binding on DNA overstretching. As dsDNA is stretched beyond its B-form contour length in 500 mM NaCl at room temperature, the force required to extend the molecule to 1.7 times its contour length is almost constant at 65 pN (black line). A model describing this transition as force-induced melting of DNA predicts that solution conditions that destabilize DNA will lower the force at which the transition occurs [24]. This is observed at high temperature (35°C, green line; [29•]), low pH (pH 3.5, orange line; [28]), high pH (pH 10.6, purple line; [28]) and low ionic strength (10 mM NaCl, red line; JR Wenner, MC Williams, I Rouzina, VA Bloomfield, unpublished data). The dashed black line shows the force/extension curve of ssDNA in 150 mM NaCl, pH 8 and room temperature [2]. The area between the solid and dashed black lines represents the helix-coil transition free energy [23]. The solid and dashed blue lines are force/extension curves for dsDNA and ssDNA, respectively, with NC [47•]. Binding of the protein lowers the helix-coil transition free energy and the cooperativity of the overstretching transition. monovalent salt dependence of DNA overstretching showed that the DNA strands must remain close together during the transition (JR Wenner, MC Williams, I Rouzina, VA Bloomfield, unpublished data). These salt dependence data are consistent with both the S-DNA and forceinduced melting models. These results are summarized in Figure 3, which shows the effect of changing solution conditions on DNA overstretching. This work has recently been published [30]. Torsionally constrained DNA In contrast to torsionally relaxed DNA, dsDNA that is not allowed to rotate freely when stretched does not exhibit an overstretching transition at 65 pN. Instead, a much less cooperative transition at a force of 110 pN is observed [31]. It has been shown that, after unwinding the DNA, the stretching curve exhibits two transitions, one at 50 pN and another at 110 pN, and, as the amount of DNA unwinding 334 Nucleic acids is increased, more of the transition occurs at 50 pN [31]. Overwinding the DNA results in an additional transition at 25 pN, which is attributed to the removal of DNA supercoiling [31]. The currently accepted model is one in which the data are interpreted as transitions between five separate forms of dsDNA [32]. However, as it is known that underwound DNA is locally denatured even at low forces [33], it seems likely that there is DNA denaturation during this transition as well, but this will require further study. In particular, a detailed study of the dependence of these transitions on solution conditions would help to explain the effect of torsional strain on DNA overstretching. Because torsional strain can build up under many physiological conditions [34], this is an important problem to solve. DNA–protein interactions from single-molecule stretching Single-molecule DNA stretching studies have been used to probe a wide range of DNA–protein interactions. These include dynamic studies, in which the action of a processive enzyme or molecular motor is directly observed as a function of time, as well as equilibrium studies, from which transition free energies have been derived. In the case of RecA, both dynamic and equilibrium properties were measured. First, the time dependence of the polymerization of RecA along a single DNA molecule was directly measured [35]. Leger et al. [36] showed that the rate of RecA binding to DNA without ATP hydrolysis increased tremendously at high forces approaching the overstretching transition. As it is known that, in the absence of ATP hydrolysis, RecA binds much more strongly to ssDNA [37], these experiments support the idea that DNA overstretching induces strand denaturation. After polymerization, the equilibrium elastic properties of RecA–DNA filaments were measured and shown to be dominated by the properties of the RecA protein [35]. Dynamic single-molecule studies of transcription and replication have directly measured polymerization velocities as a function of force, as well as the forces required to stall polymerization. [4•,38–41] In the case of RNA polymerase, although specific stall sites were identified at low forces, the average polymerization velocity was independent of tension up to forces of 25 pN, at which point transcription was reversibly stalled. In contrast, Wuite et al. [38] showed that the polymerization velocity of T7 DNA polymerase was very sensitive to tension. In addition, replication stalled at 34 pN, whereas higher forces induced fast 3′–5′ exonucleolysis. Maier et al. [4•] studied replication by the DNA polymerases Sequenase and Klenow, obtaining results similar to those of Wuite et al. [38]. Although both of these studies concluded that the polymerase must process at least two bases during each enzymatic step, a later analysis indicates that the data are also consistent with a step size of one base [42]. The kinetics and sequence dependence of DNA helicase activity by RecBCD have also been directly measured [43,44]. In these studies, the authors were able to directly observe the processive translocation of single RecBCD enzymes along single DNA molecules. The observed translocation rates were consistent with bulk measurements of DNA unwinding rates. The interaction of topoisomerase with supercoiled DNA has been directly observed using single-molecule stretching [45]. In this study, the authors were able to watch the removal of two supercoils during a single enzyme turnover. A recent study showed a direct demonstration of the forces exerted by a bacteriophage portal motor when packaging DNA [46••]. The data indicate that an internal force of about 50 pN is built up within the virus capsid when packaging the DNA. These results may shed light on the mechanism by which the virus injects DNA into cells during infection. The force-induced melting model of DNA overstretching [23] has been used to determine the free energy of the helix-coil transition from DNA overstretching. This is useful for studying DNA–protein interactions, as many proteins operate by binding to DNA and changing its stability. One such protein is HIV-1 nucleocapsid protein (NC), as was recently demonstrated by Williams et al. [47•]. NC is a nucleic acid chaperone that facilitates the rearrangement of the structure of nucleic acids in order to form the lowest energy state [48]. Until now, the mechanism of this activity was not understood. However, Williams et al. showed that NC facilitates this rearrangement by significantly lowering the cooperativity and stability of the DNA helix-coil transition (Figure 3). These results show that DNA overstretching is a powerful technique for studying proteins that may lower the helix-coil transition free energy of DNA, including other nucleic acid chaperone proteins, as well as ssDNA-binding proteins such as Escherichia coli SSB [49] and T4 gene 32 [50]. In addition, DNA-binding drugs that may stabilize or destabilize DNA could be investigated using this method. A study of anticancer drugs using AFM showed that these drugs have a significant effect on DNA overstretching [51•]. Further analysis of these results and studies of other DNA-binding drugs are needed. Conclusions Given the ability to stretch single RNA hairpin structures, as demonstrated by Liphardt et al. [17••], single-molecule force measurement techniques can be extended to studying the effect of proteins on the helix-coil transition of specific sequences and specific hairpin structures. Although both DNA overstretching and nucleic acid unzipping experiments provide a measurement of the free energy of the helix-coil transition [17••,29•], unzipping experiments allow the determination of sequence-specific information as the molecule is unzipped. Thus, the biophysics of sequence-dependent DNA- and RNA-binding proteins, such as transcription factors, could be studied in detail using this technique. In addition, single-molecule measurements of the kinetics of enzymes that operate on Force spectroscopy of single DNA and RNA molecules Williams and Rouzina 335 nucleic acids will continue to provide insights into how these molecules function. 13. Bockelmann U, Essevaz-Roulet B, Heslot F: Molecular stick-slip motion revealed by opening DNA with piconewton forces. Phys Rev Lett 1997, 79:4489-4492. Update 14. Marenduzzo D, Trovato A, Maritan A: Phase diagram of force-induced DNA unzipping in exactly solvable models. Phys Rev E 2001, 64:031901. In a recent article, single DNA molecule stretching experiments were used to study the reversible disassembly of nucleosome core particles [52•]. To do this, DNA molecules with a repeating array of histone-binding sequences were prepared. As a single DNA molecule was stretched in the presence of the histones, a repeating stretch/release pattern revealed the regular disassembly of single nucleosome core particles that were 70–80 bp in length. In addition, detailed measurements of the unzipping of λ-DNA using optical tweezers were recently published [53], as well as new measurements of the effects of DNA-binding drugs on DNA stretching curves [54]. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Zlatanova J, Lindsay SM, Leuba SH: Single molecule force spectroscopy in biology using the atomic force microscope. Prog Biophys Mol Biol 2000, 74:37-61. 2. Smith SB, Cui YJ, Bustamante C: Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 1996, 271:795-799. 3. Smith SB, Finzi L, Bustamante C: Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 1992, 258:1122-1126. 4. • Maier B, Bensimon D, Croquette V: Replication by a single DNA polymerase of a stretched single-stranded DNA. Proc Natl Acad Sci USA 2000, 97:12002-12007. In addition to direct measurements of DNA polymerase activity, the authors demonstrate the use of magnetic tweezers to obtain accurate measurements of the sequence dependence of ssDNA elasticity at low forces. 5. 6. Bouchiat C, Wang MD, Allemand JF, Strick T, Block SM, Croquette V: Estimating the persistence length of a worm-like chain molecule from force-extension measurements. Biophys J 1999, 76:409-413. Baumann CG, Smith SB, Bloomfield VA, Bustamante C: Ionic effects on the elasticity of single DNA molecules. Proc Natl Acad Sci USA 1997, 94:6185-6190. 7. • Podgornik R, Hansen PL, Parsegian VA: Elastic moduli renormalization in self-interacting stretchable polyelectrolytes. J Chem Phys 2000, 113:9343-9350. The authors present a model for the salt dependence of DNA elasticity that includes electrostatic screening between the negatively charged phosphate groups that make up the DNA backbone. 8. Bloomfield VA: DNA condensation by multivalent cations. Biopolymers 1997, 44:269-282. 9. Murayama Y, Sano M: Force measurements of a single DNA molecule in the collapsing phase transition. J Phys Soc Jpn 2000, 70:345-348. 10. Baumann CG, Bloomfield VA, Smith SB, Bustamante C, Wang MD, Block SM: Stretching of single collapsed DNA molecules. Biophys J 2000, 78:1965-1978. 11. Ali B, Amit R, Braslavsky I, Oppenheim A, Gileadi O, Stavans J: • Compaction of single DNA molecules induced by binding of integration host factor (IHF). Proc Natl Acad Sci USA 2001, 98:10658-10663. The authors present a detailed study of the effect of a DNA-binding protein on DNA bending and compaction. 12. Zhang Y, Zhou H, Ou-Yang ZC: Stretching single-stranded DNA: interplay of electrostatic, base-pairing, and base-pair stacking interactions. Biophys J 2001, 81:1133-1143. 15. Lubensky DK, Nelson DR: Pulling pinned polymers and unzipping DNA. Phys Rev Lett 2000, 85:1572-1575. 16. Cocco S, Monasson R, Marko JF: Force and kinetic barriers to unzipping of the DNA double helix. Proc Natl Acad Sci USA 2001, 98:8608-8613. 17. •• Liphardt J, Onoa B, Smith SB, Tinoco I Jr, Bustamante C: Reversible unfolding of single RNA molecules by mechanical force. Science 2001, 292:733-737. The first demonstration of RNA stretching indicates that single-molecule force spectroscopy can be used to study the structure and dynamics of RNA. 18. Zuker M: Calculating nucleic acid secondary structure. Curr Opin Struct Biol 2000, 10:303-310. 19. Cluzel P, Lebrun A, Heller C, Lavery R, Viovy JL, Chatenay D, Caron F: DNA: an extensible molecule. Science 1996, 271:792-794. 20. Konrad MW, Bolonick JI: Molecular dynamics simulation of DNA stretching is consistent with the tension observed for extension and strand separation and predicts a novel ladder structure. J Am Chem Soc 1996, 118:10989-10994. 21. Lebrun A, Lavery R: Modelling extreme stretching of DNA. Nucleic Acids Res 1996, 24:2260-2267. 22. Olson WK, Zhurkin VB: Modeling DNA deformations. Curr Opin Struct Biol 2000, 10:286-297. 23. Rouzina I, Bloomfield VA: Force-induced melting of the DNA double helix. 1. Thermodynamic analysis. Biophys J 2001, 80:882-893. 24. Rouzina I, Bloomfield VA: Force-induced melting of the DNA double helix. 2. Effect of solution conditions. Biophys J 2001, 80:894-900. 25. Rief M, Clausen-Schaumann H, Gaub HE: Sequence-dependent mechanics of single DNA molecules. Nat Struct Biol 1999, 6:346-349. 26. Clausen-Schaumann H, Rief M, Tolksdorf C, Gaub HE: Mechanical • stability of single DNA molecules. Biophys J 2000, 78:1997-2007. The authors present the first measurements of the temperature dependence of DNA overstretching and the rate dependence of DNA stretching at very high forces. 27. Evans E, Ritchie K: Dynamic strength of molecular adhesion bonds. Biophys J 1997, 72:1541-1555. 28. Williams MC, Wenner JR, Rouzina I, Bloomfield VA: The effect of pH on the overstretching transition of dsDNA: evidence of force-induced DNA melting. Biophys J 2001, 80:874-881. 29. Williams MC, Wenner JR, Rouzina I, Bloomfield VA: Entropy and • heat capacity of DNA melting from temperature dependence of single molecule stretching. Biophys J 2001, 80:1932-1939. The authors demonstrate that DNA overstretching can be used to directly measure the DNA helix-coil transition free energy at any temperature. 30. Williams MC, Rouzina I, Bloomfield VA: Thermodynamics of DNA interactions from single molecule stretching experiments. Acc Chem Res 2002, 35:159-166. 31. Leger JF, Romano G, Sarkar A, Robert J, Bourdieu L, Chatenay D, Marko JF: Structural transitions of a twisted and stretched DNA molecule. Phys Rev Lett 1999, 83:1066-1069. 32. Sarkar A, Léger J-F, Chatenay D, Marko JF: Structural transitions in DNA driven by external force and torque. Phys Rev E 2001, 63:051903. 33. Strick TR, Croquette V, Bensimon D: Homologous pairing in stretched supercoiled DNA. Proc Natl Acad Sci USA 1998, 95:10579-10583. 34. Nelson P: Transport of torsional stress in DNA. Proc Natl Acad Sci USA 1999, 96:14342-14347. 35. Hegner M, Smith SB, Bustamante C: Polymerization and mechanical properties of single RecA–DNA filaments. Proc Natl Acad Sci USA 1999, 96:10109-10114. 36. Leger JF, Robert J, Bourdieu L, Chatenay D, Marko JF: RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations. Proc Natl Acad Sci USA 1998, 95:12295-12299. 336 37. Nucleic acids McEntee K, Weinstock GM, Lehman IR: Binding of the recA protein of Escherichia coli to single- and double-stranded DNA. J Biol Chem 1981, 256:8835-8844. 38. Wuite GJ, Smith SB, Young M, Keller D, Bustamante C: Single molecule studies of the effect of template tension on T7 DNA polymerase activity. Nature 2000, 404:103-106. 39. Yin H, Wang MD, Svoboda K, Landick R, Block SM, Gelles J: Transcription against an applied force. Science 1995, 270:1653-1656. 40. Wang MD, Schnitzer MJ, Yin H, Landick R, Gelles J, Block SM: Force and velocity measured for single molecules of RNA polymerase. Science 1998, 282:902-907. 41. Davenport JR, Wuite GJ, Landick R, Bustamante C: Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase. Science 2000, 287:2497-2500. 42. Goel A, Frank-Kamenetskii MD, Ellenberger T, Herschbach D: Tuning DNA ‘strings’: modulating the rate of DNA replication with mechanical tension. Proc Natl Acad Sci USA 2001, 98:8485-8489. 43. Bianco PR, Brewer LR, Corzett M, Balhorn R, Yeh Y, Kowalczykowski SC, Baskin RJ: Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 2001, 409:374-378. 44. Dohoney KM, Gelles J: Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules. Nature 2001, 409:370-374. 45. Strick TR, Croquette V, Bensimon D: Single-molecule analysis of DNA uncoiling by a type II topoisomerase. Nature 2000, 404:901-904. 46. Smith DE, Tans SJ, Smith SB, Grimes S, Anderson DL, •• Bustamante C: The bacteriophage phi29 portal motor can package DNA against a large internal force. Nature 2001, 413:748-752. The authors present the surprising result that forces of up to 50 pN can be used to package DNA into a virus. These results suggest that internal pressure may provide the driving force for the injection of viral DNA into a host cell. 47. • Williams MC, Rouzina I, Wenner JR, Gorelick RJ, Musier-Forsyth K, Bloomfield VA: Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching. Proc Natl Acad Sci USA 2001, 98:6121-6126. Single DNA molecule stretching was used to investigate the interaction between DNA and a nucleic-acid-binding protein essential for HIV replication. These results indicate that the zinc finger structures of the protein are essential to its capability to alter the helix-coil transition of nucleic acids, and that this capability is required for a crucial step in the reverse transcription process during HIV replication. 48. Rein A, Henderson LE, Levin JG: Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication. Trends Biochem Sci 1998, 23:297-301. 49. 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