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Single-molecule studies of nucleic acid motors
Single-molecule studies of nucleic acid motors Ralf Seidel1 and Cees Dekker2 Nucleic acid motors comprise a variety of structurally, mechanistically and functionally very different enzymes. These motor proteins have in common the ability to directionally move DNA or RNA, or to move along DNA or RNA using a chemical energy source such as ATP. Recently, it became possible to study the action of a single motor on single DNA or RNA molecules in real time; this has provided unprecedented insight into the behavior and mechanism of these motors. As a result, the past few years have witnessed an enormous increase in such single-molecule studies of a variety of different motor systems. Particular highlights have included the investigation of the sequence-dependent behavior and helical tracking of motors, and the attainment of the ultimate (i.e. single base pair) resolution, which enables the detection of individual single base motor steps. Addresses 1 Biotechnological Centre, University of Technology Dresden, Tatzberg 47-51, 01307 Dresden, Germany 2 Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands Corresponding author: Dekker, Cees ([email protected])
Current Opinion in Structural Biology 2007, 17:80–86 This review comes from a themed issue on Protein–nucleic acid interactions Edited by James M Berger and Christoph W Mu¨ller Available online 5th January 2007 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.12.003
Introduction Nucleic acid motors are ubiquitous in all fundamental cellular processes that involve DNA or RNA, for example, transcription, replication, translation, packaging and maintenance of chromosome structure, restriction, DNA repair and bacterial conjugation. Most motor proteins use ATP or other nucleotide triphosphates (NTPs) to move along nucleic acids. Very often, such a simple translocation process is accompanied by another nucleic acid remodeling function. For example, RNA polymerase moves along the DNA, reads out its sequence and transcribes it into RNA. Helicases unzip or unwind duplex DNA to allow other proteins to access the DNA strands. To understand the function and mechanism of these enzymes, it would be greatly beneficial to observe a single enzyme moving along DNA or RNA at atomic resolution in real time. This, however, is currently Current Opinion in Structural Biology 2007, 17:80–86
impossible. On one hand, crystal structure data provide the necessary spatial resolution, but crystallographic methods only afford snapshots of individual states of proteins, that is, the dynamics of the process is lacking. On the other hand, classical biochemical analyses provide dynamic information, but average over large molecular ensembles. Single-molecule techniques allow the observation of the dynamic action of a single motor protein on a DNA or RNA molecule with a resolution that is now better than the 0.34 nm of single base pair steps [1]. Single-molecule experiments also provide unique access to parameters such as the forces produced by motors and offer an excellent complement to classical techniques to obtain an understanding of motor function at the atomic level. This review summarizes the achievements of singlemolecule methods in the field of nucleic acid motors during the past two years. In particular, the direct observation of single motor steps, the study of DNA helix tracking, the investigation of the sequence-dependent behavior of motor movement and the unraveling of the mechanochemistry of single motors will be discussed. Furthermore, a perspective will be given on future challenges and possible developments for the field.
How to measure translocation of a single motor Most assays currently used to observe the action of nucleic acid motors start with the stretching of DNA or RNA molecules using an external force that is applied by a hydrodynamic flow or optical or magnetic tweezers (see Figure 1a). Motor activity is then measured either as a change in the DNA or RNA length or directly from the position of a labeled motor on the stretched DNA or RNA (Figure 1b). Other single-molecule methods, such as fluorescence resonance energy transfer (FRET; see [2]), have also been applied to motor proteins; however, to date, they have not been used to directly measure translocation. Optical tweezers, the preferred method of choice so far, provide currently the highest spatial and temporal resolution, whereas magnetic tweezers conveniently allow supercoils to be introduced by applying torque to the DNA and enable measurements to be made at extremely low constant forces (as low as 0.01 pN). Hydrodynamic flow, on the other hand, is technically the least complicated method. To transform motor movement along DNA into a change in the measured length of the stretched nucleic acid structure or the position of a marker bound to the motor, several strategies have been followed depending on the www.sciencedirect.com
Single-molecule studies of nucleic acid motors Seidel and Dekker 81
Figure 1
Single-molecule methods for measuring translocation of nucleic acid motors. (a) Most methods rely on stretched DNA or RNA. Stretching can be achieved using: (i) a laser trap, which can pull on a molecule that is attached between a surface and a bead; (ii) a magnetic field, which exerts force on a superparamagnetic bead; or (iii) a hydrodynamic flow, which acts on the DNA or RNA molecule itself (top) or on a bead attached to the molecule (bottom). (b) Motor movement on a stretched nucleic acid molecule can be detected in different ways. (i) Immobilizing the motor and detecting the shortening or lengthening of the DNA or RNA molecule. (ii) Motors that translocate DNA or RNA into loops directly shorten the molecule; this can be detected. (iii) For motors that convert ssDNA to dsDNA or dsDNA to ssDNA (such as DNA polymerases or helicases), a change in the molecule length can be detected, because ssDNA and dsDNA have different extension depending on the stretching force. (iv) Direct observation of a translocating motor on stretched DNA or RNA. Visualization of the motor is achieved by labeling it with (fluorescent) beads or fluorescent antibodies or by simple visual detection of protein aggregates. (v) Motors such as RuvAB, which specifically move a homologous four-way junction, can be detected when DNA is pumped from two branches of a given orientation into the perpendicularly oriented branches. (vi) Unzipping of a hairpin by a helicase. Unzipping each base pair increases the length of the ssDNA or ssRNA part by two bases. Red arrows symbolize the direction of DNA movement, black arrows the direction of the applied force, F.
actual motor system (Figure 1b): (i) immobilization of the motor at the surface of a glass slide or a bead held by a micropipette or an optical trap [1,3,4,5,6,7]; (ii) monitoring the change in length as a result of DNA loop formation during translocation [8,9–11,12,13–16]; (iii) using the different force-dependent extensions of dsDNA and ssDNA [17]; (iv) direct observation of motor movement along a stretched DNA molecule by detecting protein aggregates [13,14] or fluorescent beads or antibodies that are attached to the motor [14,18,19]; (v) using branch migration of a homologous four-way junction [20,21]; and (vi) measuring the unzipping of a hairpin by a helicase [22]. The use of one of these methods can powerfully extract relevant motor properties at the single-molecule level. For example, in the assay shown in Figure 1b(iv), one can track a particle attached to the motor. In principle, this is now possible at an 1 nm resolution [23]. Following the particle over time directly yields the velocity and typical www.sciencedirect.com
processivity (translocation length) of a single motor on DNA. Typical values are given in Table 1. A wide range of parameters can be noted, in line with the varying needs of the different cellular functions of the motors (Box 1).
Resolving single motor steps The ultimate goal in detecting motor movement is to resolve individual motor steps or substeps, as they provide interesting insight into motor function and mechanism. Whereas the steps of cytoskeletal motors, most famously the 8.2 nm steps of kinesin on microtubules [24], were resolved long ago, similar experiments on nucleic acid motors remained challenging, as their steps are on the order of a single or a few base pairs. Recently, however, the 0.3 nm single base pair detection limit was broken using sophisticated ultrastable optical tweezers [1] and it became possible to detect the single base pair stepping motion of RNA polymerase on DNA (Figure 2). This now opens the way to answering mechanistic questions regarding the motor stepping motion and resolving Current Opinion in Structural Biology 2007, 17:80–86
82 Protein–nucleic acid interactions
Table 1 Properties of nucleic acid motors characterized using single-molecule techniques during the past two yearsa. Name
Type
Function
No. catalytic subunits
Velocity (bp s1) b
Force (pN) c
Processivity (bp) d
Step size (bp)
Helix tracking
References
E. coli RNA polymerase T7 RNA polymerase FtsK
RNA polymerase
Transcription
1
16
25
Several kbp
1
Yes
[1,3,4]
RNA polymerase
Transcription
1
130
16
>1000
1
Yes
[5]
dsDNA translocase
6
5000
40
>5000
2 or 13
No
[12,13–16]
F29 portal motor RuvAB
dsDNA translocase
Chromosome segregation Viral packaging
5
100
57
15 000
NN
NN
[6]
6
43
25
4000
NN
NN
[20,21,33]
HCV NS3 RNA helicase EcoR124I
RNA helicase
Migrates Holliday junctions HCV replication
1 or 2
50
NN
18
11
NN
[22]
dsDNA translocase
1
550
>5
5000
1–2
Yes
[8,9,10]
RSC complex
dsDNA translocase
1
350
>2
400
12
No
[11]
Rad54
dsDNA translocase
NN
300
NN
12 000
NN
NN
[18]
RecBCD
DNA helicase
2
520
8
30 000
<6 or 23
Yes
[19]
B. subtilis DNA uptake T7 replisome
ssDNA translocase
Type I restriction enzyme Chromatin remodeling Homologous recombination dsDNA break processing Horizontal gene transfer DNA unwinding and synthesis
NN
80
45
>10 000
NN
NN
[7]
6 and 1
160
NN
17 000
NA
Yes
[17]
a b c d
dsDNA translocase
DNA replicase
Most of the data were measured at room temperature. Velocity indicates the mean velocity at saturating fuel conditions. The force is the maximum force determined to date. The processivity is the mean translocated distance in the absence of force. NA, not applicable; NN, not known.
the sequence-dependent behavior of motors at single base pair resolution (see below; [4]). Another example is the NS3 RNA helicase from hepatitis C virus (HCV), for which individual motor steps have been detected [22]. This helicase shows 11 bp steps with smaller, 2–5 bp, substeps. A ‘quantum inchworm’ model [25] has been proposed, whereby a translocator domain carries out one big step and a helix opener domain subsequently unwinds the RNA with multiple small steps. How this Box 1 About forces, energies and steps What force can a DNA motor produce? Movement of DNA motors is typically driven by hydrolysis of NTPs. For ATP hydrolysis, for example, the gain in free energy, including enthalpic and entropic contributions, is, under cellular concentrations of ATP, ADP and Pi (1 mM, 10 mM and 1 mM, respectively), about 100 1021 J (or alternatively 100 pNnm). This is about 25 kBT, where 1 kBT (4.1 pNnm) is the energy scale of thermal fluctuations. For kinesin, with a step size of 8.2 nm, the gain in energy from ATP hydrolysis would therefore be enough to generate a maximum force of 12 pN. Experimentally, maximum forces of about 5 pN were measured [34]. In contrast to the cytoskeletal motors, DNA motors can generate much higher forces because of their much smaller step sizes of one or a few base pairs. A motor with a 1 bp step size (0.34 nm) could, in principle, generate 300 pN. The strongest nucleic acid motor reported to date is the F29 viral packaging motor, which can generate almost 60 pN of force [35]. Note that the DNA double helix breaks down at a stretching force of about 65 pN [36].
Current Opinion in Structural Biology 2007, 17:80–86
model translates into a structural interpretation (the enzyme is about the same size as the large step) remains to be resolved.
Coupling DNA supercoiling to translocation How do motor proteins move along DNA? Specifically, do they simply move along it in a linear fashion or do they spiral around it, tracking the helical pitch? The latter would set certain constraints regarding the accumulation of supercoils (Figure 3). The question is almost impossible to address in bulk biochemical experiments. The first demonstration of helix tracking was shown for RNA polymerase [26], which can drive the rotation of a magnetic bead if the enzyme is attached to a glass substrate. To correlate translocation distance with the number of induced rotations, the translocation speed was determined in bulk measurements. Magnetic tweezers can be applied to directly correlate — in one single-molecule assay — the translocation distance and the number of induced rotations [8,11,12]. The amount of supercoiling can be determined from the length reduction, which is caused by the formation of plectonemes (Figure 3). Starting from negatively supercoiled DNA, an enzyme motor (e.g. a loop-forming motor) that introduces positive supercoils into the stretched DNA will deplete the applied negative supercoils, thus www.sciencedirect.com
Single-molecule studies of nucleic acid motors Seidel and Dekker 83
Figure 2
Detection of single base pair stepping by E. coli RNA polymerase [1]. (a) A single RNA polymerase (RNAP; green) is attached to a bead held in a weak optical trap (right). It is also attached via the upstream DNA to a bead held in a strong optical trap (left). Movement of RNA polymerase causes the weakly trapped bead to move to the right, which is detected. The weak trap is operated with the bead near the peak of the force/extension curve, which provides a passive force clamp. F determines the force and x the bead position. The white arrow indicates the movement of the bead away from its equilibrium position, caused by the translocating RNA polymerase. (b) Representative record of single base pair steps of RNA polymerase. Figure reproduced with permission from S Block.
dissolving the plectonemes. As a result, the DNA length increases. At maximum length increase, all the previously induced supercoils are removed. At this position, the DNA length is only shortened because of the loop, which was pulled in by the motor (Figure 3b, at about 30 s). By comparing this position with the original DNA length before supercoiling (see Figure 3b, at about 10 s), the translocated distance can be calculated. Using this method, it was established, for the first time, that the motor unit of the type I restriction enzyme EcoR124I induces one supercoil per helical pitch, that is to say, it tracks directly along the helical pitch of the DNA [8]. This fits additional observations that the enzyme is a dsDNA translocase that moves along the 30 to 50 strand with a step size much smaller than the helical pitch [9]. For the hexameric motor protein FtsK, however, the same technique demonstrated that the motor induces only 0.07 supercoils per translocated helical pitch. This can be explained by a step size that is slightly larger than the helical pitch [12]. Alternatively, and in better agreement with the enzyme dimensions determined using X-ray crystallography, a similar low number (0.14) of supercoils per translocated helical pitch is obtained if each of the six monomers moves the DNA about 2 bp [27]. Interestingly, a similar value of 0.04–0.15 supercoils per translocated helical pitch has been determined for the RSC chromatin remodeling complex [11], although this complex contains a single motor subunit and belongs to the same superfamily of helicases as EcoR124I. www.sciencedirect.com
Sequence information from studies of single motors Detection of the position of a motor on its nucleic acid track enables the study of motor action at special sequences that affect motor activity, such as pausing sites for RNA polymerase [28,29] and the x hot spot for RecBCD [19,30]. The development of an ultrastable trap [1] with significant resolution improvements has now enabled studies of the pausing of RNA polymerase with base pair accuracy. This led to the identification of the sequences of pausing sites [4]. Furthermore, by lowering one of the four NTP concentrations, artificial pauses can be generated at a specific base. Carrying out this experiment for all four NTPs and aligning the four pause patterns enabled the direct sequencing of a single DNA molecule over a length of about 30 bp [3]. In the case of FtsK, the bacterial motor required for chromosome segregation during cell division, single-molecule analysis has contributed to the identification of special sequences recognized by the motor. FtsK aligns the two so-called dif sites, targets for the site-specific XerC and XerD recombinases, at the division septum between the two dividing cells and sorts the chromosomal DNA to the daughter cells. FtsK has to translocate towards dif, but not much beyond that. Indeed, by observing small protein aggregates on a piece of chromosomal DNA around dif, it was found that FtsK can continuously translocate towards dif, but its translocation direction is reversed after passing and translocating away from dif [13]. The so-called KOPS [15] or FRS [14] Current Opinion in Structural Biology 2007, 17:80–86
84 Protein–nucleic acid interactions
Figure 3
Generation of supercoils by a helix-tracking motor. (a) A free helix-tracking motor will swivel around the DNA with one rotation per helix translocated (left). If constraints are placed on its ability to swivel, the motor would translocate, but not change the number of helical turns between top and bottom ahead of and behind the motor. Therefore, the shortening DNA ahead of the motor becomes increasingly positively supercoiled, whereas the growing region of DNA behind the motor becomes increasingly negatively supercoiled. A similar situation is found for loop-extruding DNA translocases, whereby the loop produced by the motor is negatively supercoiled and the DNA ahead of the motor is positively supercoiled. (b) EcoR124I is an example of a DNA-looping motor protein [8]. In a magnetic tweezers setup, DNA is coiled to 20 supercoils, which induces DNA shortening due to plectoneme formation. At 28 s, the EcoR124I motor acts on the DNA. Generation of positive supercoils by the motor removes the plectonemes, which causes the DNA length to increase. At maximum length, that is, after complete removal of the negative supercoils, the translocated distance can be determined. For EcoR124I, a value of 11 2 bp per translocated DNA per generated supercoil has been determined, which indicates helix tracking by EcoR124I. Adapted from [8].
sequences are responsible for FtsK reversal. The singlemolecule experiments were used to identify sequence stretches that contain translocation reversal sites; this helped to discard other KOPS/FRS candidate sequences determined from bioinformatics analysis [14].
Ratchet or power stroke? A unique feature of single-molecule techniques is that they enable direct assessment of the forces generated by motors. Measuring force/velocity relationships as a function of ATP concentration can be extremely helpful to understand the mechanochemistry of a single cycle of ATP hydrolysis [31]. This allows, for example, the identification of the hydrolysis cycle step during which forcedependent forward movement occurs. In two independent studies of Escherichia coli RNA polymerase [1] and bacteriophage T7 RNA polymerase [5], analysis of NTPdependent force/velocity relationships strongly supported a Brownian ratchet model for forward movement, whereby the polymerase fluctuates between a pre- and a post-translocated state. NTP hydrolysis and product release then lock the polymerase irreversibly into the post-translocated state. A power-stroke model, whereby translocation is directly irreversibly driven by product Current Opinion in Structural Biology 2007, 17:80–86
(pyrophosphate) release, was found to be inconsistent with the measured data. Similar experiments have also been carried out for the packaging motor of Bacillus subtilis phage F29 [6]. This ring-like motor consists of five ATPase subunits, which encircle and translocate dsDNA in a similar manner to FtsK. In this case, it was found that translocation does not occur during ATP binding, but rather upon phosphate release, and that the motor subunits do not act independently, but in a coordinated consecutive manner.
Coordination of different motors in complex machines In most cellular processes, nucleic acid motors do not act in isolation, but rather function in a coordinated fashion with other motor and non-motor proteins. Because motor properties such as velocity can change upon the interaction of multiple motors [32], it is of interest to investigate the interplay of motors with other proteins. This has been done for the replisome of bacteriophage T7 [17]. At a replication fork, several motors have to be coordinated — the helicase, the lagging-strand primase and the DNA polymerases. This could potentially be problematic as primases synthesize RNA primers at a www.sciencedirect.com
Single-molecule studies of nucleic acid motors Seidel and Dekker 85
rate that is orders of magnitude lower than the rate of DNA synthesis by DNA polymerases at the fork, that is, DNA leading-strand synthesis would outpace laggingstrand synthesis. Single-molecule analysis, however, revealed that the primase acts as a molecular break and stops fork progression during primer synthesis to ensure coordination of DNA synthesis at both strands [17].
An ultrastable optical trap was developed and used to directly detect single base pair steps of RNA polymerase. This led to the establishment of a Brownian ratchet mechanism for E. coli RNA polymerase.
Future perspectives
4.
What are the challenges for single-molecule studies of nucleic acid motors? Clearly, more high-resolution studies are required to understand how the large variety of cellular motors convert chemical energy into force-producing forward movement, revealing step sizes, possible helix tracking and the mechanochemistry. For example, it would be very interesting to examine the coordination of the multiple ATPase subunits of hexameric motors such as FtsK and the F29 portal motor. A combination of tweezers experiments and single-molecule fluorescence techniques could be helpful in this context. Beyond detailed mechanistic questions, it will become increasingly important in the future to understand motor function in situations that more closely reflect in vivo conditions or, even better, to probe single motor proteins within a single cell. First steps in this direction have been made by in vitro studies of the whole T7 replisome [17] and in vivo studies of DNA uptake by B. subtilis [7]. Single-molecule techniques can potentially contribute to a variety of fields. For example, motor action is very important in the context of chromatin. Chromatin remodeling enzymes such as the RSC complex [11] and Rad54 [18] have unambiguously been identified as DNA motors using single-molecule methods. How do their motor properties change if these studies are carried out with chromatin rather than with bare DNA, and what is the mechanism of these enzymes in chromatin remodeling? Another interesting field will be to understand (i.e. visualize) the interplay of the enzyme machinery involved in eukaryotic homologous recombination.
2.
Myong S, Stevens BC, Ha T: Bridging conformational dynamics and function using single-molecule spectroscopy. Structure 2006, 14:633-643.
3.
Greenleaf WJ, Block SM: Single-molecule, motion-based DNA sequencing using RNA polymerase. Science 2006, 313:801.
Herbert KM, La Porta A, Wong BJ, Mooney RA, Neuman KC, Landick R, Block SM: Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 2006, 125:1083-1094. The authors detected and identified RNA polymerase pausing sequences at base pair resolution.
5.
Thomen P, Lopez PJ, Heslot F: Unravelling the mechanism of RNA-polymerase forward motion by using mechanical force. Phys Rev Lett 2005, 94:128102. A Brownian ratchet mechanism for T7 RNA polymerase was established. 6.
Chemla YR, Aathavan K, Michaelis J, Grimes S, Jardine PJ, Anderson DL, Bustamante C: Mechanism of force generation of a viral DNA packaging motor. Cell 2005, 122:683-692.
7.
Maier B, Chen I, Dubnau D, Sheetz MP: DNA transport into Bacillus subtilis requires proton motive force to generate large molecular forces. Nat Struct Mol Biol 2004, 11:643-649.
8.
Seidel R, van Noort J, van der Scheer C, Bloom JGP, Dekker NH, Dutta CF, Blundell A, Robinson T, Firman K, Dekker C: Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I. Nat Struct Mol Biol 2004, 11:838-843. The first report of the direct coupling of supercoil generation and translocation for a DNA motor using a single-molecule experiment. It was demonstrated that the dsDNA motor EcoR124I translocates the helical pitch of the DNA. 9.
Stanley LK, Seidel R, van der Scheer C, Dekker NH, Szczelkun MD, Dekker C: When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124l. EMBO J 2006, 25:2230-2239.
10. Seidel R, Bloom JGP, van Noort J, Dutta CF, Dekker NH, Firman K, Szczelkun MD, Dekker C: Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I. EMBO J 2005, 24:4188-4197. 11. Lia G, Praly E, Ferreira H, Stockdale C, Tse-Dinh YC, Dunlap D, Croquette V, Bensimon D, Owen-Hughes T: Direct observation of DNA distortion by the RSC complex. Mol Cell 2006, 21:417-425. 12. Saleh OA, Bigot S, Barre FX, Allemand JF: Analysis of DNA supercoil induction by FtsK indicates translocation without groove-tracking. Nat Struct Mol Biol 2005, 12:436-440. The first identification of a dsDNA motor that does not translocate along the helical pitch of the DNA.
It is clear that we can expect plenty of further exciting single-molecule results concerning nucleic acid motors.
13. Pease PJ, Levy O, Cost GJ, Gore J, Ptacin JL, Sherratt D, Bustamante C, Cozzarelli NR: Sequence-directed DNA translocation by purified FtsK. Science 2005, 307:586-590.
Acknowledgements
14. Levy O, Ptacin JL, Pease PJ, Gore J, Eisen MB, Bustamante C, Cozzarelli NR: Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase. Proc Natl Acad Sci USA 2005, 102:17618-17623.
The authors thank MD Szczelkun for carefully reading the manuscript. RS acknowledges support by the Deutsche Forschungsgemeinschaft (DFG). CD acknowledges support by FOM, NWO, NanoNed and Bionano-switch.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM: Direct observation of base-pair stepping by RNA polymerase. Nature 2005, 438:460-465.
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15. Bigot S, Saleh OA, Lesterlin C, Pages C, El Karoui M, Dennis C, Grigoriev M, Allemand JF, Barre FX, Cornet F: KOPS: DNA motifs that control E-coli chromosome segregation by orienting the FtsK translocase. EMBO J 2005, 24:3770-3780. 16. Saleh OA, Perals C, Barre FX, Allemand JF: Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment. EMBO J 2004, 23:2430-2439. 17. Lee JB, Hite RK, Hamdan SM, Xie XS, Richardson CC, van Oijen AM: DNA primase acts as a molecular brake in DNA replication. Nature 2006, 439:621-624. The real-time observation of the coordination of multiple DNA motors at the T7 replication fork was described. Current Opinion in Structural Biology 2007, 17:80–86
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18. Amitani I, Baskin RJ, Kowalczykowski SC: Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol Cell 2006, 23:143-148.
27. Massey TH, Mercogliano CP, Yates J, Sherratt DJ, Lowe J: Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol Cell 2006, 23:457-469.
19. Handa N, Bianco PR, Baskin RJ, Kowalczykowski SC: Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition. Mol Cell 2005, 17:745-750.
28. Shaevitz JW, Abbondanzieri EA, Landick R, Block SM: Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 2003, 426:684-687.
20. Dawid A, Croquette V, Grigoriev M, Heslot F: Singlemolecule study of RuvAB-mediated Holliday-junction migration. Proc Natl Acad Sci USA 2004, 101:11611-11616. 21. Amit R, Gileadi O, Stavans J: Direct observation of RuvABcatalyzed branch migration of single Holliday junctions. Proc Natl Acad Sci USA 2004, 101:11605-11610. 22. Dumont S, Cheng W, Serebrov V, Beran RK, Tinoco I, Pyle AM, Bustamante C: RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 2006, 439:105-108. The direct detection of the motor steps of a helicase was reported. The RNA helicase HCV NS3 moves with large 11 bp steps and smaller substeps of 2–5 bp.
29. Neuman KC, Abbondanzieri EA, Landick R, Gelles J, Block SM: Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 2003, 115:437-447. 30. Spies M, Bianco PR, Dillingham MS, Handa N, Baskin RJ, Kowalczykowski SC: A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase. Cell 2003, 114:647-654. 31. Keller D, Bustamante C: The mechanochemistry of molecular motors. Biophys J 2000, 78:541-556. 32. Stano NM, Jeong YJ, Donmez I, Tummalapalli P, Levin MK, Patel SS: DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 2005, 435:370-373.
23. Gordon MP, Ha T, Selvin PR: Single-molecule high-resolution imaging with photobleaching. Proc Natl Acad Sci USA 2004, 101:6462-6465.
33. Han YW, Tani T, Hayashi M, Hishida T, Iwasaki H, Shinagawa H, Harada Y: Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvARuvB protein complex. Proc Natl Acad Sci USA 2006, 103:11544-11548.
24. Svoboda K, Schmidt CF, Schnapp BJ, Block SM: Direct observation of kinesin stepping by optical trapping interferometry. Nature 1993, 365:721-727.
34. Howard J (Ed): Mechanics of Motor Proteins and the Cytoskeleton. Sinauer Associates; 2001.
25. Bianco PR, Kowalczykowski SC: Translocation step size and mechanism of the RecBC DNA helicase. Nature 2000, 405:368-372. 26. Harada Y, Ohara O, Takatsuki A, Itoh H, Shimamoto N, Kinosita K: Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase. Nature 2001, 409:113-115.
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35. Smith DE, Tans SJ, Smith SB, Grimes S, Anderson DL, Bustamante C: The bacteriophage phi 29 portal motor can package DNA against a large internal force. Nature 2001, 413:748-752. 36. Smith SB, Cui YJ, Bustamante C: Overstretching B-DNA: the elastic response of individual double-stranded and singlestranded DNA molecules. Science 1996, 271:795-799.
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Current Opinion in Structural Biology 2007, 17:80–86 This review comes from a themed issue on Protein–nucleic acid interactions Edited by James M Berger and Christoph W Mu¨ller Available online 5th January 2007 0959-440X/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2006.12.003
Introduction Nucleic acid motors are ubiquitous in all fundamental cellular processes that involve DNA or RNA, for example, transcription, replication, translation, packaging and maintenance of chromosome structure, restriction, DNA repair and bacterial conjugation. Most motor proteins use ATP or other nucleotide triphosphates (NTPs) to move along nucleic acids. Very often, such a simple translocation process is accompanied by another nucleic acid remodeling function. For example, RNA polymerase moves along the DNA, reads out its sequence and transcribes it into RNA. Helicases unzip or unwind duplex DNA to allow other proteins to access the DNA strands. To understand the function and mechanism of these enzymes, it would be greatly beneficial to observe a single enzyme moving along DNA or RNA at atomic resolution in real time. This, however, is currently Current Opinion in Structural Biology 2007, 17:80–86
impossible. On one hand, crystal structure data provide the necessary spatial resolution, but crystallographic methods only afford snapshots of individual states of proteins, that is, the dynamics of the process is lacking. On the other hand, classical biochemical analyses provide dynamic information, but average over large molecular ensembles. Single-molecule techniques allow the observation of the dynamic action of a single motor protein on a DNA or RNA molecule with a resolution that is now better than the 0.34 nm of single base pair steps [1]. Single-molecule experiments also provide unique access to parameters such as the forces produced by motors and offer an excellent complement to classical techniques to obtain an understanding of motor function at the atomic level. This review summarizes the achievements of singlemolecule methods in the field of nucleic acid motors during the past two years. In particular, the direct observation of single motor steps, the study of DNA helix tracking, the investigation of the sequence-dependent behavior of motor movement and the unraveling of the mechanochemistry of single motors will be discussed. Furthermore, a perspective will be given on future challenges and possible developments for the field.
How to measure translocation of a single motor Most assays currently used to observe the action of nucleic acid motors start with the stretching of DNA or RNA molecules using an external force that is applied by a hydrodynamic flow or optical or magnetic tweezers (see Figure 1a). Motor activity is then measured either as a change in the DNA or RNA length or directly from the position of a labeled motor on the stretched DNA or RNA (Figure 1b). Other single-molecule methods, such as fluorescence resonance energy transfer (FRET; see [2]), have also been applied to motor proteins; however, to date, they have not been used to directly measure translocation. Optical tweezers, the preferred method of choice so far, provide currently the highest spatial and temporal resolution, whereas magnetic tweezers conveniently allow supercoils to be introduced by applying torque to the DNA and enable measurements to be made at extremely low constant forces (as low as 0.01 pN). Hydrodynamic flow, on the other hand, is technically the least complicated method. To transform motor movement along DNA into a change in the measured length of the stretched nucleic acid structure or the position of a marker bound to the motor, several strategies have been followed depending on the www.sciencedirect.com
Single-molecule studies of nucleic acid motors Seidel and Dekker 81
Figure 1
Single-molecule methods for measuring translocation of nucleic acid motors. (a) Most methods rely on stretched DNA or RNA. Stretching can be achieved using: (i) a laser trap, which can pull on a molecule that is attached between a surface and a bead; (ii) a magnetic field, which exerts force on a superparamagnetic bead; or (iii) a hydrodynamic flow, which acts on the DNA or RNA molecule itself (top) or on a bead attached to the molecule (bottom). (b) Motor movement on a stretched nucleic acid molecule can be detected in different ways. (i) Immobilizing the motor and detecting the shortening or lengthening of the DNA or RNA molecule. (ii) Motors that translocate DNA or RNA into loops directly shorten the molecule; this can be detected. (iii) For motors that convert ssDNA to dsDNA or dsDNA to ssDNA (such as DNA polymerases or helicases), a change in the molecule length can be detected, because ssDNA and dsDNA have different extension depending on the stretching force. (iv) Direct observation of a translocating motor on stretched DNA or RNA. Visualization of the motor is achieved by labeling it with (fluorescent) beads or fluorescent antibodies or by simple visual detection of protein aggregates. (v) Motors such as RuvAB, which specifically move a homologous four-way junction, can be detected when DNA is pumped from two branches of a given orientation into the perpendicularly oriented branches. (vi) Unzipping of a hairpin by a helicase. Unzipping each base pair increases the length of the ssDNA or ssRNA part by two bases. Red arrows symbolize the direction of DNA movement, black arrows the direction of the applied force, F.
actual motor system (Figure 1b): (i) immobilization of the motor at the surface of a glass slide or a bead held by a micropipette or an optical trap [1,3,4,5,6,7]; (ii) monitoring the change in length as a result of DNA loop formation during translocation [8,9–11,12,13–16]; (iii) using the different force-dependent extensions of dsDNA and ssDNA [17]; (iv) direct observation of motor movement along a stretched DNA molecule by detecting protein aggregates [13,14] or fluorescent beads or antibodies that are attached to the motor [14,18,19]; (v) using branch migration of a homologous four-way junction [20,21]; and (vi) measuring the unzipping of a hairpin by a helicase [22]. The use of one of these methods can powerfully extract relevant motor properties at the single-molecule level. For example, in the assay shown in Figure 1b(iv), one can track a particle attached to the motor. In principle, this is now possible at an 1 nm resolution [23]. Following the particle over time directly yields the velocity and typical www.sciencedirect.com
processivity (translocation length) of a single motor on DNA. Typical values are given in Table 1. A wide range of parameters can be noted, in line with the varying needs of the different cellular functions of the motors (Box 1).
Resolving single motor steps The ultimate goal in detecting motor movement is to resolve individual motor steps or substeps, as they provide interesting insight into motor function and mechanism. Whereas the steps of cytoskeletal motors, most famously the 8.2 nm steps of kinesin on microtubules [24], were resolved long ago, similar experiments on nucleic acid motors remained challenging, as their steps are on the order of a single or a few base pairs. Recently, however, the 0.3 nm single base pair detection limit was broken using sophisticated ultrastable optical tweezers [1] and it became possible to detect the single base pair stepping motion of RNA polymerase on DNA (Figure 2). This now opens the way to answering mechanistic questions regarding the motor stepping motion and resolving Current Opinion in Structural Biology 2007, 17:80–86
82 Protein–nucleic acid interactions
Table 1 Properties of nucleic acid motors characterized using single-molecule techniques during the past two yearsa. Name
Type
Function
No. catalytic subunits
Velocity (bp s1) b
Force (pN) c
Processivity (bp) d
Step size (bp)
Helix tracking
References
E. coli RNA polymerase T7 RNA polymerase FtsK
RNA polymerase
Transcription
1
16
25
Several kbp
1
Yes
[1,3,4]
RNA polymerase
Transcription
1
130
16
>1000
1
Yes
[5]
dsDNA translocase
6
5000
40
>5000
2 or 13
No
[12,13–16]
F29 portal motor RuvAB
dsDNA translocase
Chromosome segregation Viral packaging
5
100
57
15 000
NN
NN
[6]
6
43
25
4000
NN
NN
[20,21,33]
HCV NS3 RNA helicase EcoR124I
RNA helicase
Migrates Holliday junctions HCV replication
1 or 2
50
NN
18
11
NN
[22]
dsDNA translocase
1
550
>5
5000
1–2
Yes
[8,9,10]
RSC complex
dsDNA translocase
1
350
>2
400
12
No
[11]
Rad54
dsDNA translocase
NN
300
NN
12 000
NN
NN
[18]
RecBCD
DNA helicase
2
520
8
30 000
<6 or 23
Yes
[19]
B. subtilis DNA uptake T7 replisome
ssDNA translocase
Type I restriction enzyme Chromatin remodeling Homologous recombination dsDNA break processing Horizontal gene transfer DNA unwinding and synthesis
NN
80
45
>10 000
NN
NN
[7]
6 and 1
160
NN
17 000
NA
Yes
[17]
a b c d
dsDNA translocase
DNA replicase
Most of the data were measured at room temperature. Velocity indicates the mean velocity at saturating fuel conditions. The force is the maximum force determined to date. The processivity is the mean translocated distance in the absence of force. NA, not applicable; NN, not known.
the sequence-dependent behavior of motors at single base pair resolution (see below; [4]). Another example is the NS3 RNA helicase from hepatitis C virus (HCV), for which individual motor steps have been detected [22]. This helicase shows 11 bp steps with smaller, 2–5 bp, substeps. A ‘quantum inchworm’ model [25] has been proposed, whereby a translocator domain carries out one big step and a helix opener domain subsequently unwinds the RNA with multiple small steps. How this Box 1 About forces, energies and steps What force can a DNA motor produce? Movement of DNA motors is typically driven by hydrolysis of NTPs. For ATP hydrolysis, for example, the gain in free energy, including enthalpic and entropic contributions, is, under cellular concentrations of ATP, ADP and Pi (1 mM, 10 mM and 1 mM, respectively), about 100 1021 J (or alternatively 100 pNnm). This is about 25 kBT, where 1 kBT (4.1 pNnm) is the energy scale of thermal fluctuations. For kinesin, with a step size of 8.2 nm, the gain in energy from ATP hydrolysis would therefore be enough to generate a maximum force of 12 pN. Experimentally, maximum forces of about 5 pN were measured [34]. In contrast to the cytoskeletal motors, DNA motors can generate much higher forces because of their much smaller step sizes of one or a few base pairs. A motor with a 1 bp step size (0.34 nm) could, in principle, generate 300 pN. The strongest nucleic acid motor reported to date is the F29 viral packaging motor, which can generate almost 60 pN of force [35]. Note that the DNA double helix breaks down at a stretching force of about 65 pN [36].
Current Opinion in Structural Biology 2007, 17:80–86
model translates into a structural interpretation (the enzyme is about the same size as the large step) remains to be resolved.
Coupling DNA supercoiling to translocation How do motor proteins move along DNA? Specifically, do they simply move along it in a linear fashion or do they spiral around it, tracking the helical pitch? The latter would set certain constraints regarding the accumulation of supercoils (Figure 3). The question is almost impossible to address in bulk biochemical experiments. The first demonstration of helix tracking was shown for RNA polymerase [26], which can drive the rotation of a magnetic bead if the enzyme is attached to a glass substrate. To correlate translocation distance with the number of induced rotations, the translocation speed was determined in bulk measurements. Magnetic tweezers can be applied to directly correlate — in one single-molecule assay — the translocation distance and the number of induced rotations [8,11,12]. The amount of supercoiling can be determined from the length reduction, which is caused by the formation of plectonemes (Figure 3). Starting from negatively supercoiled DNA, an enzyme motor (e.g. a loop-forming motor) that introduces positive supercoils into the stretched DNA will deplete the applied negative supercoils, thus www.sciencedirect.com
Single-molecule studies of nucleic acid motors Seidel and Dekker 83
Figure 2
Detection of single base pair stepping by E. coli RNA polymerase [1]. (a) A single RNA polymerase (RNAP; green) is attached to a bead held in a weak optical trap (right). It is also attached via the upstream DNA to a bead held in a strong optical trap (left). Movement of RNA polymerase causes the weakly trapped bead to move to the right, which is detected. The weak trap is operated with the bead near the peak of the force/extension curve, which provides a passive force clamp. F determines the force and x the bead position. The white arrow indicates the movement of the bead away from its equilibrium position, caused by the translocating RNA polymerase. (b) Representative record of single base pair steps of RNA polymerase. Figure reproduced with permission from S Block.
dissolving the plectonemes. As a result, the DNA length increases. At maximum length increase, all the previously induced supercoils are removed. At this position, the DNA length is only shortened because of the loop, which was pulled in by the motor (Figure 3b, at about 30 s). By comparing this position with the original DNA length before supercoiling (see Figure 3b, at about 10 s), the translocated distance can be calculated. Using this method, it was established, for the first time, that the motor unit of the type I restriction enzyme EcoR124I induces one supercoil per helical pitch, that is to say, it tracks directly along the helical pitch of the DNA [8]. This fits additional observations that the enzyme is a dsDNA translocase that moves along the 30 to 50 strand with a step size much smaller than the helical pitch [9]. For the hexameric motor protein FtsK, however, the same technique demonstrated that the motor induces only 0.07 supercoils per translocated helical pitch. This can be explained by a step size that is slightly larger than the helical pitch [12]. Alternatively, and in better agreement with the enzyme dimensions determined using X-ray crystallography, a similar low number (0.14) of supercoils per translocated helical pitch is obtained if each of the six monomers moves the DNA about 2 bp [27]. Interestingly, a similar value of 0.04–0.15 supercoils per translocated helical pitch has been determined for the RSC chromatin remodeling complex [11], although this complex contains a single motor subunit and belongs to the same superfamily of helicases as EcoR124I. www.sciencedirect.com
Sequence information from studies of single motors Detection of the position of a motor on its nucleic acid track enables the study of motor action at special sequences that affect motor activity, such as pausing sites for RNA polymerase [28,29] and the x hot spot for RecBCD [19,30]. The development of an ultrastable trap [1] with significant resolution improvements has now enabled studies of the pausing of RNA polymerase with base pair accuracy. This led to the identification of the sequences of pausing sites [4]. Furthermore, by lowering one of the four NTP concentrations, artificial pauses can be generated at a specific base. Carrying out this experiment for all four NTPs and aligning the four pause patterns enabled the direct sequencing of a single DNA molecule over a length of about 30 bp [3]. In the case of FtsK, the bacterial motor required for chromosome segregation during cell division, single-molecule analysis has contributed to the identification of special sequences recognized by the motor. FtsK aligns the two so-called dif sites, targets for the site-specific XerC and XerD recombinases, at the division septum between the two dividing cells and sorts the chromosomal DNA to the daughter cells. FtsK has to translocate towards dif, but not much beyond that. Indeed, by observing small protein aggregates on a piece of chromosomal DNA around dif, it was found that FtsK can continuously translocate towards dif, but its translocation direction is reversed after passing and translocating away from dif [13]. The so-called KOPS [15] or FRS [14] Current Opinion in Structural Biology 2007, 17:80–86
84 Protein–nucleic acid interactions
Figure 3
Generation of supercoils by a helix-tracking motor. (a) A free helix-tracking motor will swivel around the DNA with one rotation per helix translocated (left). If constraints are placed on its ability to swivel, the motor would translocate, but not change the number of helical turns between top and bottom ahead of and behind the motor. Therefore, the shortening DNA ahead of the motor becomes increasingly positively supercoiled, whereas the growing region of DNA behind the motor becomes increasingly negatively supercoiled. A similar situation is found for loop-extruding DNA translocases, whereby the loop produced by the motor is negatively supercoiled and the DNA ahead of the motor is positively supercoiled. (b) EcoR124I is an example of a DNA-looping motor protein [8]. In a magnetic tweezers setup, DNA is coiled to 20 supercoils, which induces DNA shortening due to plectoneme formation. At 28 s, the EcoR124I motor acts on the DNA. Generation of positive supercoils by the motor removes the plectonemes, which causes the DNA length to increase. At maximum length, that is, after complete removal of the negative supercoils, the translocated distance can be determined. For EcoR124I, a value of 11 2 bp per translocated DNA per generated supercoil has been determined, which indicates helix tracking by EcoR124I. Adapted from [8].
sequences are responsible for FtsK reversal. The singlemolecule experiments were used to identify sequence stretches that contain translocation reversal sites; this helped to discard other KOPS/FRS candidate sequences determined from bioinformatics analysis [14].
Ratchet or power stroke? A unique feature of single-molecule techniques is that they enable direct assessment of the forces generated by motors. Measuring force/velocity relationships as a function of ATP concentration can be extremely helpful to understand the mechanochemistry of a single cycle of ATP hydrolysis [31]. This allows, for example, the identification of the hydrolysis cycle step during which forcedependent forward movement occurs. In two independent studies of Escherichia coli RNA polymerase [1] and bacteriophage T7 RNA polymerase [5], analysis of NTPdependent force/velocity relationships strongly supported a Brownian ratchet model for forward movement, whereby the polymerase fluctuates between a pre- and a post-translocated state. NTP hydrolysis and product release then lock the polymerase irreversibly into the post-translocated state. A power-stroke model, whereby translocation is directly irreversibly driven by product Current Opinion in Structural Biology 2007, 17:80–86
(pyrophosphate) release, was found to be inconsistent with the measured data. Similar experiments have also been carried out for the packaging motor of Bacillus subtilis phage F29 [6]. This ring-like motor consists of five ATPase subunits, which encircle and translocate dsDNA in a similar manner to FtsK. In this case, it was found that translocation does not occur during ATP binding, but rather upon phosphate release, and that the motor subunits do not act independently, but in a coordinated consecutive manner.
Coordination of different motors in complex machines In most cellular processes, nucleic acid motors do not act in isolation, but rather function in a coordinated fashion with other motor and non-motor proteins. Because motor properties such as velocity can change upon the interaction of multiple motors [32], it is of interest to investigate the interplay of motors with other proteins. This has been done for the replisome of bacteriophage T7 [17]. At a replication fork, several motors have to be coordinated — the helicase, the lagging-strand primase and the DNA polymerases. This could potentially be problematic as primases synthesize RNA primers at a www.sciencedirect.com
Single-molecule studies of nucleic acid motors Seidel and Dekker 85
rate that is orders of magnitude lower than the rate of DNA synthesis by DNA polymerases at the fork, that is, DNA leading-strand synthesis would outpace laggingstrand synthesis. Single-molecule analysis, however, revealed that the primase acts as a molecular break and stops fork progression during primer synthesis to ensure coordination of DNA synthesis at both strands [17].
An ultrastable optical trap was developed and used to directly detect single base pair steps of RNA polymerase. This led to the establishment of a Brownian ratchet mechanism for E. coli RNA polymerase.
Future perspectives
4.
What are the challenges for single-molecule studies of nucleic acid motors? Clearly, more high-resolution studies are required to understand how the large variety of cellular motors convert chemical energy into force-producing forward movement, revealing step sizes, possible helix tracking and the mechanochemistry. For example, it would be very interesting to examine the coordination of the multiple ATPase subunits of hexameric motors such as FtsK and the F29 portal motor. A combination of tweezers experiments and single-molecule fluorescence techniques could be helpful in this context. Beyond detailed mechanistic questions, it will become increasingly important in the future to understand motor function in situations that more closely reflect in vivo conditions or, even better, to probe single motor proteins within a single cell. First steps in this direction have been made by in vitro studies of the whole T7 replisome [17] and in vivo studies of DNA uptake by B. subtilis [7]. Single-molecule techniques can potentially contribute to a variety of fields. For example, motor action is very important in the context of chromatin. Chromatin remodeling enzymes such as the RSC complex [11] and Rad54 [18] have unambiguously been identified as DNA motors using single-molecule methods. How do their motor properties change if these studies are carried out with chromatin rather than with bare DNA, and what is the mechanism of these enzymes in chromatin remodeling? Another interesting field will be to understand (i.e. visualize) the interplay of the enzyme machinery involved in eukaryotic homologous recombination.
2.
Myong S, Stevens BC, Ha T: Bridging conformational dynamics and function using single-molecule spectroscopy. Structure 2006, 14:633-643.
3.
Greenleaf WJ, Block SM: Single-molecule, motion-based DNA sequencing using RNA polymerase. Science 2006, 313:801.
Herbert KM, La Porta A, Wong BJ, Mooney RA, Neuman KC, Landick R, Block SM: Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 2006, 125:1083-1094. The authors detected and identified RNA polymerase pausing sequences at base pair resolution.
5.
Thomen P, Lopez PJ, Heslot F: Unravelling the mechanism of RNA-polymerase forward motion by using mechanical force. Phys Rev Lett 2005, 94:128102. A Brownian ratchet mechanism for T7 RNA polymerase was established. 6.
Chemla YR, Aathavan K, Michaelis J, Grimes S, Jardine PJ, Anderson DL, Bustamante C: Mechanism of force generation of a viral DNA packaging motor. Cell 2005, 122:683-692.
7.
Maier B, Chen I, Dubnau D, Sheetz MP: DNA transport into Bacillus subtilis requires proton motive force to generate large molecular forces. Nat Struct Mol Biol 2004, 11:643-649.
8.
Seidel R, van Noort J, van der Scheer C, Bloom JGP, Dekker NH, Dutta CF, Blundell A, Robinson T, Firman K, Dekker C: Real-time observation of DNA translocation by the type I restriction modification enzyme EcoR124I. Nat Struct Mol Biol 2004, 11:838-843. The first report of the direct coupling of supercoil generation and translocation for a DNA motor using a single-molecule experiment. It was demonstrated that the dsDNA motor EcoR124I translocates the helical pitch of the DNA. 9.
Stanley LK, Seidel R, van der Scheer C, Dekker NH, Szczelkun MD, Dekker C: When a helicase is not a helicase: dsDNA tracking by the motor protein EcoR124l. EMBO J 2006, 25:2230-2239.
10. Seidel R, Bloom JGP, van Noort J, Dutta CF, Dekker NH, Firman K, Szczelkun MD, Dekker C: Dynamics of initiation, termination and reinitiation of DNA translocation by the motor protein EcoR124I. EMBO J 2005, 24:4188-4197. 11. Lia G, Praly E, Ferreira H, Stockdale C, Tse-Dinh YC, Dunlap D, Croquette V, Bensimon D, Owen-Hughes T: Direct observation of DNA distortion by the RSC complex. Mol Cell 2006, 21:417-425. 12. Saleh OA, Bigot S, Barre FX, Allemand JF: Analysis of DNA supercoil induction by FtsK indicates translocation without groove-tracking. Nat Struct Mol Biol 2005, 12:436-440. The first identification of a dsDNA motor that does not translocate along the helical pitch of the DNA.
It is clear that we can expect plenty of further exciting single-molecule results concerning nucleic acid motors.
13. Pease PJ, Levy O, Cost GJ, Gore J, Ptacin JL, Sherratt D, Bustamante C, Cozzarelli NR: Sequence-directed DNA translocation by purified FtsK. Science 2005, 307:586-590.
Acknowledgements
14. Levy O, Ptacin JL, Pease PJ, Gore J, Eisen MB, Bustamante C, Cozzarelli NR: Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase. Proc Natl Acad Sci USA 2005, 102:17618-17623.
The authors thank MD Szczelkun for carefully reading the manuscript. RS acknowledges support by the Deutsche Forschungsgemeinschaft (DFG). CD acknowledges support by FOM, NWO, NanoNed and Bionano-switch.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM: Direct observation of base-pair stepping by RNA polymerase. Nature 2005, 438:460-465.
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15. Bigot S, Saleh OA, Lesterlin C, Pages C, El Karoui M, Dennis C, Grigoriev M, Allemand JF, Barre FX, Cornet F: KOPS: DNA motifs that control E-coli chromosome segregation by orienting the FtsK translocase. EMBO J 2005, 24:3770-3780. 16. Saleh OA, Perals C, Barre FX, Allemand JF: Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment. EMBO J 2004, 23:2430-2439. 17. Lee JB, Hite RK, Hamdan SM, Xie XS, Richardson CC, van Oijen AM: DNA primase acts as a molecular brake in DNA replication. Nature 2006, 439:621-624. The real-time observation of the coordination of multiple DNA motors at the T7 replication fork was described. Current Opinion in Structural Biology 2007, 17:80–86
86 Protein–nucleic acid interactions
18. Amitani I, Baskin RJ, Kowalczykowski SC: Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol Cell 2006, 23:143-148.
27. Massey TH, Mercogliano CP, Yates J, Sherratt DJ, Lowe J: Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol Cell 2006, 23:457-469.
19. Handa N, Bianco PR, Baskin RJ, Kowalczykowski SC: Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition. Mol Cell 2005, 17:745-750.
28. Shaevitz JW, Abbondanzieri EA, Landick R, Block SM: Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 2003, 426:684-687.
20. Dawid A, Croquette V, Grigoriev M, Heslot F: Singlemolecule study of RuvAB-mediated Holliday-junction migration. Proc Natl Acad Sci USA 2004, 101:11611-11616. 21. Amit R, Gileadi O, Stavans J: Direct observation of RuvABcatalyzed branch migration of single Holliday junctions. Proc Natl Acad Sci USA 2004, 101:11605-11610. 22. Dumont S, Cheng W, Serebrov V, Beran RK, Tinoco I, Pyle AM, Bustamante C: RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 2006, 439:105-108. The direct detection of the motor steps of a helicase was reported. The RNA helicase HCV NS3 moves with large 11 bp steps and smaller substeps of 2–5 bp.
29. Neuman KC, Abbondanzieri EA, Landick R, Gelles J, Block SM: Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 2003, 115:437-447. 30. Spies M, Bianco PR, Dillingham MS, Handa N, Baskin RJ, Kowalczykowski SC: A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase. Cell 2003, 114:647-654. 31. Keller D, Bustamante C: The mechanochemistry of molecular motors. Biophys J 2000, 78:541-556. 32. Stano NM, Jeong YJ, Donmez I, Tummalapalli P, Levin MK, Patel SS: DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 2005, 435:370-373.
23. Gordon MP, Ha T, Selvin PR: Single-molecule high-resolution imaging with photobleaching. Proc Natl Acad Sci USA 2004, 101:6462-6465.
33. Han YW, Tani T, Hayashi M, Hishida T, Iwasaki H, Shinagawa H, Harada Y: Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvARuvB protein complex. Proc Natl Acad Sci USA 2006, 103:11544-11548.
24. Svoboda K, Schmidt CF, Schnapp BJ, Block SM: Direct observation of kinesin stepping by optical trapping interferometry. Nature 1993, 365:721-727.
34. Howard J (Ed): Mechanics of Motor Proteins and the Cytoskeleton. Sinauer Associates; 2001.
25. Bianco PR, Kowalczykowski SC: Translocation step size and mechanism of the RecBC DNA helicase. Nature 2000, 405:368-372. 26. Harada Y, Ohara O, Takatsuki A, Itoh H, Shimamoto N, Kinosita K: Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase. Nature 2001, 409:113-115.
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35. Smith DE, Tans SJ, Smith SB, Grimes S, Anderson DL, Bustamante C: The bacteriophage phi 29 portal motor can package DNA against a large internal force. Nature 2001, 413:748-752. 36. Smith SB, Cui YJ, Bustamante C: Overstretching B-DNA: the elastic response of individual double-stranded and singlestranded DNA molecules. Science 1996, 271:795-799.
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