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Fast, DNA‐sequence independent translocation by FtsK in a single‐molecule experiment

Fast, DNA‐sequence independent translocation by FtsK in a single‐molecule experiment The EMBO Journal (2004) 23, 2430–2439 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment 1 2 such, they are thought to form hexameric motors, which are Omar A Saleh , Corine Pe´rals , 2, responsible for the active translocation process. They are Franc¸ois-Xavier Barre * and 1,3, involved in a wide range of functions: TraSA is encoded by Jean-Franc¸ois Allemand * the plasmid pSAM2 of Streptomyces ambofaciens,and is Laboratoire de Physique Statistique et Departement de Biologie, Ecole 2 responsible for the transfer of the plasmid from donor cells Normale Superieure, Paris, France, Laboratoire de Microbiologie et into acceptor cells during conjugation (Possoz et al, 2001). ´ ´ ´ de Genetique Moleculaire, Toulouse, France and Laboratoire Pasteur, ´ ´ Departement de Chimie, Ecole Normale Superieure, Paris, France SpoIIIE is responsible for the transport of a chromosome into the small polar prespore compartment during sporulation in Escherichia coli FtsK is an essential cell division protein, Bacillus subtilis (Bath et al, 2000). FtsK is an essential which is thought to pump chromosomal DNA through the Escherichia coli cell division protein (Begg et al, 1995). Its closing septum in an oriented manner by following DNA N-terminal membrane domain is localized to the division sequence polarity. Here, we perform single-molecule mea- septum (Wang and Lutkenhaus, 1998; Yu et al, 1998a), and is surements of translocation by FtsK , a derivative that 50C necessary for its formation (Draper et al, 1998; Chen and functions as a DNA translocase in vitro. FtsK transloca- Beckwith, 2001). Its C-terminal motor domain (FtsK )is 50C tion follows Michaelis–Menten kinetics, with a maximum implicated in chromosome segregation (Liu et al, 1998; Yu speed of B6.7 kbp/s. We present results on the effect of et al, 1998b). Indeed, FtsK has two essential roles in applied force on the speed, distance translocated, and the chromosome dimer resolution (see Aussel et al, 2002; mean times during and between protein activity. Capiaux et al, 2002; Corre and Louarn, 2002, and references Surprisingly, we observe that FtsK can spontaneously 50C therein for review). Chromosome dimers, formed by homo- reverse its translocation direction on a fragment of E. coli logous recombination in organisms with circular chromo- chromosomal DNA, indicating that DNA sequence is not somes, are a threat to normal repartitioning of genetic the sole determinant of translocation direction. We con- information at cell division. In E. coli, chromosome dimers clude that in vivo polarization of FtsK translocation could are resolved into monomers by the addition of a crossover at require the presence of cofactors; alternatively, we propose a specific 28 bp site, dif, by two tyrosine recombinases, XerC a model in which tension in the DNA directs FtsK translo- and XerD. Chromosome dimer resolution first requires colo- cation. calization of the dif sites; this is accomplished by transloca- The EMBO Journal (2004) 23, 2430–2439. doi:10.1038/ tion of the chromosomes by FtsK (Capiaux et al, 2002; Corre sj.emboj.7600242; Published online 27 May 2004 and Louarn, 2002). Secondly, the recombination reaction Subject Categories: genome stability & dynamics; itself is activated by FtsK (Aussel et al, 2002), probably microbiology & pathogens through a direct interaction with XerC/D (Yates et al, 2003; Keywords: chromosome segregation; DNA translocation; Massey et al, 2004). In addition, DNA translocation by FtsK FtsK; magnetic tweezers; molecular motor could participate in segregation of normal chromosomes (Yu et al, 1998b; Donachie, 2002; Lau et al, 2003). Coupled to these processes, there must be a mechanism of translocation polarity; that is, an orienting mechanism that Introduction ensures the DNA is transported in the correct direction. In B. subtilis, it has been argued that preferential assembly of Active transportation of DNA from one cellular compartment SpoIIIE in one daughter cell establishes polarity in chromo- to another is central to many biological processes, such as some partitioning during sporulation (Sharp and Pogliano, viral DNA packaging, conjugation, sporulation, and cell divi- 2002). In normal vegetative growth in E. coli, in contrast to sion. In bacteria, a new class of proteins was recently sporulating B. subtilis, there is no obvious morphological nor identified that mediate the transport of double-stranded protein expression difference between the two daughter cells DNA through cell membranes and cell walls: the FtsK/ that could explain asymmetric assembly of FtsK. Instead, it SpoIIIE/TraSA family (Bath et al, 2000; Possoz et al, 2001; has been postulated that FtsK translocation polarity is defined Aussel et al, 2002). These proteins belong to the AAAþ by chromosomal sequence polarity, that is, a high skew of (ATPase Associated with various Activities) superfamily. As oligomeric sequences along the two replichores that inverts at *Corresponding authors. Franc¸ois-Xavier Barre, Laboratoire de dif (Salzberg et al, 1998; Lobry and Louarn, 2003). Microbiologie et de Ge´ne´tique Mole´culaire, 118 Route de Narbonne, Chromosome dimer resolution is effective only when dif is 31062 Toulouse, France. Tel.: þ 33 5 61 33 59 86; located within a narrow zone of the chromosome (Cornet Fax: þ 33 5 61 33 58 86; E-mail: [email protected] or Jean-Franc¸ois et al, 1996), functionally defined as the junction between Allemand, Laboratoire de Physique Statistique, Ecole Normale Supe´rieure, 24, Rue Lhomond, 75005 Paris, France. DNA segments of opposite sequence polarity (Corre et al, Tel.: þ33 144323496; Fax: þ33 144323433; 2000; Perals et al, 2000). Sequence polarity can be perturbed E-mail: [email protected] by the introduction, close to dif, of exogenous DNA, such as the genome of phage lambda (Corre et al, 2000). This change Received: 23 February 2004; accepted: 27 April 2004; published online: 27 May 2004 inactivates chromosome dimer resolution, and augments the 2430 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al probability of endogenous recombination in an FtsK-depen- Results dent manner (Corre and Louarn, 2002). This has led to the FtsK causes transient decreases of DNA extension 50C idea that polarization of the chromosome sequence could Magnetic beads (4.5 mm) are tethered to a glass capillary by a direct FtsK’s translocation so as to juxtapose the chromoso- section of DNA (see Figure 1A), as described in Materials and mal dif sites; this mechanism could also assist in clearing the methods. We use a portion of lambda DNA in all experiments septum of any residual DNA. presented, with one exception (noted below) in which we use In vitro evidence for DNA translocation by the ATPase- motor domains of SpoIIIE and FtsK has been obtained, but all the assays employed were indirect, precluding detailed ana- lysis of the translocation reaction (Bath et al, 2000; Aussel et al, 2002; Ip et al, 2003). Here, we directly monitor the DNA translocase activity of FtsK , an active, oligomeric C-term- 50C inal truncation of FtsK (Aussel et al, 2002), at the single- molecule level using magnetic tweezers (Strick et al, 1996). We measure, in various conditions of applied force and ATP concentration, the velocity, length, and duration of individual translocation events. Using these data, we deduce basic mechanochemical parameters of the protein’s translocation reaction. Surprisingly, we find that FtsK can spontaneously re- 50C verse its translocation direction, and thus travel both ways on the same segment of DNA. We test translocation on both lambda genomic DNA and an E. coli chromosomal fragment that has been shown to be polarized in vivo (Perals et al, 2000). No bias in translocation is observed on either of these substrates, suggesting that DNA sequence polarity does not directly affect FtsK activity. We conclude that in vivo 50C polarization of FtsK translocation could require additional cofactors, such as domains of the full protein not included in the truncation used here. Alternatively, we propose a me- chanism in which force, rather than DNA sequence, directs the protein’s in vivo motion. Figure 1 (A) Schematic representation of the measurement appa- ratus. A magnetic bead is tethered to a glass capillary by a single, nicked DNA molecule. Magnets above the capillary create a field gradient that pulls on the bead with a force F, which is determined by measuring the bead’s lateral fluctuations dx. By optically track- ing the bead, we measure the extension L of the DNA with time. (B) Typical measurement of DNA extension, both with and without FtsK in the solution. The plotted data were measured at 50C F¼ 10.7 pN and with 5 mM ATP; the data sets have been offset for clarity. We attribute the transient decreases in DNA extension to the creation of loops of DNA by single complexes of FtsK .(C)A 50C typical individual event extracted from the data shown in (B). All events begin with a constant-velocity decrease in DNA extension; we attribute the slope of the descent to the translocation velocity of the complex. The velocity, distance travelled d, and on-time are easily measured because of the well-defined event shape. As sketched, loop formation requires the complex to bind the DNA in two locations; see Figure 6 and Discussion for details. (D) FtsK 50C activity versus ATP concentration for both bulk (rate of ATP consumption; red curve) and single molecule (translocation velo- city; green points) assays. Both data sets are well fit by the Michaelis–Menten equation V [ATP]/([ATP]þ K ), with, respec- max m tively, V ¼ 3072 ATP/s and 6.770.1 kbp/s, and K ¼ 270740 max m and 330720 mM. The fit to the single-molecule data (blue curve) is shown; it is highly coincident with the bulk data fit, which is omitted for clarity. Single-molecule data are taken at FB5 pN, and each point is the average of typically 100 events; plotted bars indicate standard error. Inset: Histogram of measured translocation velocities (open circles) with best-fit Gaussian curve (solid line); data taken at 5 mM ATP and F¼ 6 pN. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2431 | | Single-molecule studies of FtsK translocation OA Saleh et al a fragment of E. coli chromosomal DNA. We search for beads presence of DNA (Aussel et al, 2002), are consistent with a that are tethered by a single, nicked DNA molecule; while we mechanism in which ATP consumption is restricted to DNA- are able to identify and utilize unnicked DNA molecules, we bound FtsK , and leads directly to DNA translocation. 50C concentrate here on nicked molecules so as to avoid the topological complications of supercoiling. Once a suitable Reversal of the translocation direction bead is found, we flush the capillary with ATP-laden buffer, After the initial translocation, the recovery back to the add purified FtsK monomers to a final concentration of original bead height occurs in one of two ways, as shown 50C 5–10 nM, and begin tracking the DNA extension with time. in Figure 2A. The first type of recovery is an abrupt jump that After a short time, many transient decreases in DNA exten- occurs at speeds 420 mm/s, which is too fast to be resolved sion are observed, as shown in Figure 1B. Preliminary data on in our system. This is consistent with the protein completely unnicked molecules (OA Saleh, S Bigot, FX Barre and JF unbinding, and the bead rising at a rate determined by the Allemand, in preparation) are in general very similar to applied force and hydrodynamic Stokes drag (e.g. a 2 pN nicked-molecule data; thus, nicks appear not to affect the force will move a 4.5 mm diameter bead through water at a protein activity. The typical waiting time between events terminal velocity of B50 mm/s). The second type of recovery (B5 s) is greater than the typical event timespan (B0.2 s), is a slower, constant-velocity increase, with a slope (at low indicating that the measured activity is due to the action of a forces) nearly equivalent to that of the decrease. We believe single protein complex. Note that the structure of FtsK in that slow recoveries are caused by a single complex that, after 50C solution is unknown, and apparently multimeric (Aussel et al, decreasing the bead height, reverses its translocation direc- 2002); throughout the text, we refer to the active protein unit tion and causes the bead to rise (see Discussion and Figure 2A as a complex. The events continue to occur for a long time sketches). Both types of recoveries occur at all forces; at a without need of additional proteins or ATP. We detect no given force, events with slow and fast recoveries are ran- events in the absence of either ATP or FtsK , and we find domly interspersed. However, it is clear that the frequency of 50C that existing activity stops upon addition of EDTA to a final occurrence of direction reversals depends on the applied concentration of 20 mM. From these observations, we con- force: the proportion of slow recoveries decreases from clude that the events are caused by the ATP-dependent DNA B50% at low forces to nearly zero at high forces (inset, translocation activity of FtsK . Figure 2A). Rarely, we observe events with more complicated 50C mixed recoveries, wherein the bead height first increases slowly, then abruptly, or vice versa (see Supplementary data). Translocation velocity represents protein activity Direction reversal has potential implications for the in vivo Each event begins with a linear decrease in bead height (Figure 1C), from which we can extract both the translocation mechanism of FtsK translocation polarity, as it shows that, on velocity and the total distance travelled by the protein. Very the section of lambda DNA utilized, the protein can travel in rarely, we observe events containing pauses that interrupt the both directions on the same DNA sequence. To further constant-velocity decrease. The pauses have no correlation explore this effect, we perform measurements on a fragment of E. coli chromosomal DNA with a proven in vivo polarity with position along the DNA, and only occur in situations effect (Perals et al, 2000). As shown in Figure 2B, we still where many protein complexes appear to be active. We attribute pausing to protein–protein interactions, and do not observe direction reversal events on the E. coli DNA, with no characterize them here; see Supplementary data for more difference in either the shape of individual events, or the information. Within a single measurement (i.e., on one DNA frequency of occurrence of slow recoveries. Furthermore, full- molecule, and at constant force and ATP concentration), the length direction reversal events occur (wherein the bead is translocation velocity measured from all events is normally brought from full height down to the capillary surface, then back up again, all at constant speed; see Figure 2B), indicat- distributed (inset, Figure 1D). Infrequently, an event is ob- ing that no subsection of the E. coli DNA fragment has a served with almost precisely double the mean translocation velocity of other events in the data set (see Supplementary definitive effect on the translocation direction of the complex. data). Such events presumably arise from two-motor activity (see Discussion), and are removed from all the analysis Force dependence of the translocation velocity presented. The width of the distribution is accounted for by In our assay, the applied force F depends only on the distance measurement noise and variations due to the stochastic of the magnets to the capillary; we can easily adjust this stepping motion of the motor protein (Svoboda et al, 1994); distance to study the dependence of translocation velocity on thus, within the experimental noise, we observe no protein- force. During initial translocation, the protein lowers the to-protein or sequence-based velocity variation. bead, and must perform work against the applied force. In To confirm that FtsK ’s ATPase activity causes its trans- contrast, during direction-reversed translocation, the bead 50C location, we compare, at various ATP concentrations, the rises, so the protein works with the (upward) force. single-molecule velocity with the bulk ATPase activity mea- Thus, we can use initial translocation to measure the sured in an excess of DNA (insuring each functional protein is velocity with forces hindering the protein’s motion, and active). As shown in Figure 1D, the assays agree quite well: reversed translocation to measure the velocity with each independently indicates that FtsK follows simple forces assisting the motion (following previous practice, we 50C Michaelis–Menten kinetics with K B0.3 mM ATP and respec- define a hindering force as positive). In Figure 3, we show tive maximum velocities of B6.7 kbp/s and B30 ATP/ the force–velocity relationships measured in the presence of 1 FtsK monomer/s. Note that the latter is only a relative and 5 mM ATP. At both concentrations, there is a clear 50C measure since it ignores the presence of nonfunctional (mis- plateau of constant velocity for small forces, along with a folded) proteins. These results, in the light of previous data transition to increasingly higher velocities at large assisting showing that ATPase activity is highly stimulated in the forces; this transition occurs, respectively, at 4 and 11 pN 2432 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al F < 0 F > 0 Assisting Hindering force force 5 mM ATP 1 mM ATP Figure 3 Force–velocity relations for FtsK measured at 1 and 50C 5 mM ATP. At both ATP concentrations, there is a plateau of constant velocity for small forces. The velocity measured at 5 mM ATP begins to decrease above a hindering force of 15 pN, while no decrease is seen in the 1 mM data up to 29 pN. The hindering-force data indicate that the FtsK reaction pathway has force-indepen- 50C dent biochemical step(s) in series with force-dependent mechanical step(s) (see Discussion). Plotted bars indicate standard error; large error estimates indicate regimes where the event frequency and size are small (see Figure 4). Mean event length and on-time decrease with force We are unable to measure the velocity above B35 pN because of a strong decrease in the size of each event, and in the frequency of event occurrence. The length and duration (i.e., on-time) of each event correspond, respectively, to the distance travelled by the protein, and the time spent bound to the DNA, before unbinding or reversing direction (see Figure 1C). Within a measurement at a given force and ATP concentration, both distance and on-time vary from event to event. We find that the distribution of each is always exponential (see inset, Figure 2 (A) Illustration of different types of recoveries. After Figure 4B), indicating a constant unbinding probability decreasing, the DNA extension can recover its full length either with time (and thus distance, given the nearly constant abruptly (black curve) or slowly (red curve). As sketched, we velocity within a given measurement; see inset, Figure 1D); attribute the former to protein unbinding, and the latter to a reversal of the translocation direction. The fraction of events exhibiting a we can then fit an exponential curve to each distribution and slow recovery is force dependent, as plotted in the inset. The extract the mean distance and on-time for the given condi- distance travelled before a reversal of direction is random, and tions. In this way, we measure, at constant 5 mM ATP, the consistent with the distribution of distances travelled before un- variation of mean distance and on-time with applied force. As binding (see Figure 4). The events plotted were measured at 18 pN and 5 mM ATP, and are offset for clarity. The inset includes data shown in Figure 4A, the mean on-time decreases strongly, and taken at ATP concentrations from 0.5 to 5 mM; we observe no exponentially, with force. Fitting the data to t exp(F/F ) 0 0 variation in the slow fraction with ATP. (B) Translocation of a gives a decay constant F ¼11.370.9 pN and a mean on-time fragment of E. coli chromosomal DNA that has been shown to at zero force of t ¼1.670.3 s. The mean distance travelled have in vivo polar activity (Perals et al, 2000); data taken at 5 mM ATP and F¼ 1 pN. All the three plotted events have slow, direction- also decreases strongly with force, as shown in Figure 4B. reversal recoveries. In particular, in the first event, FtsK translo- 50C At low forces, the translocation velocity is fairly constant cates the entire length of the DNA in both directions, indicating that (Figure 3), so we expect the mean distance to vary in the no portion of the DNA sequence has a strong effect in biasing the same manner as the mean on-time. This is indeed the case: translocation direction. an exponential fit to the low-force points gives a decay constant F ¼11.673.2 pN, nearly equivalent to the decay for 1 and 5 mM ATP. At 5 mM ATP, the velocity begins to constant of the mean on-time. Above 20 pN, the mean clearly decrease for F415 pN; no such transition is seen in distance drops off more quickly due to the decrease in the 1 mM data up to F ¼ 29 pN. velocity at high forces. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2433 | | / Single-molecule studies of FtsK translocation OA Saleh et al Force dependence of the off-times Along with the distances and on-times, we extract from each measurement distributions of the off-times, that is, the time duration between events (see Figure 1B). The off-time dis- tributions from a given measurement clearly do not follow a single exponential curve, but rather a curve that is the sum of (at least) two exponentials with, respectively, fast and slow time constants (see Supplementary data). The presence of Off-times fast and slow rebinding rates is presumably due to the varying timescales of processes that contribute to protein– DNA binding (see Discussion). We find strong variations in On-times the mean off-time for different measurements, which we F = 11.3 ± 0.9 pN attribute to variations in the amount and active percentage of the added protein. However, since a single addition of protein to the capillary results in long-lasting activity, we can vary the force and confidently measure the response of the off-times at constant protein concentration. As shown in Figure 4A, we find that the mean values of the off-time increase with force, indicating that binding rate of the protein to the DNA is decreased by the application of higher forces. Noise analysis reveals FtsK step size 50C The completion of an enzymatic cycle results in a single forward step of a motor protein. If the step size is larger than Exponential fit, the measurement noise, and the average time to complete an F = 11.6 ± 3.2 pN enzymatic cycle is larger than the time resolution of the measurement, it would be possible to observe individual steps directly. This is not the case here: we observe no discrete steps in the traces of bead position versus time. However, it is still possible to estimate the step size by considering the fluctuations in the measurement. In the absence of protein activity, the resolution of our measure- ment is limited by the bead’s Brownian fluctuations, which, at low frequency f, create a frequency-independent ‘white’ noise. During protein activity (i.e., during the linear decrease in bead height), we find that the measurement noise in- creases at low frequencies as 1/f , and thus cannot be attributed only to Brownian fluctuations (see Figure 5). Such a low-frequency increase in noise has been observed in previous single-molecule measurements (Svoboda et al, 1994), and has been shown to be proportional to the enzy- matic step size (Svoboda et al, 1994; Charvin et al, 2002); it is Figure 4 (A) Force dependence of the on- and off-times at 5 mM caused by the random distribution of the times between ATP. As with the distance travelled (see inset, B), the distribution of individual steps (Svoboda et al, 1994). on-times from many events within a measurement is exponential To estimate the step size, we select the active segment of a (data not shown), indicating a constant unbinding probability with time. We extract a mean on-time from each distribution through suitable event and compute, at each time point, the difference fitting an exponential function. Here, we plot the variation with between the bead’s measured position and its mean position force of the mean on-time (circles), and fit it with an exponential (as predicted by the mean velocity). We then find the power function t exp(F/F ). The mean on-time decreases with force with 0 0 spectrum of that difference, average the spectra over many a decay constant F ¼11.370.9 pN and an on-time at zero force of t ¼1.670.3 s. This exponential force dependence can be simply 0 events from a single measurement, and fit this average with explained if we assume that binding of the protein causes a the theoretically predicted curve (Charvin et al, 2002); an distortion that decreases the DNA length by at least example is shown in Figure 5. By performing this analysis, we 0.3670.03 nm (see Discussion). (B) Dependence of the mean estimate that the step size of FtsK is 1272 bp. In our distance travelled per event on force at 5 mM ATP. The mean 50C distance travelled at a given force should follow directly from the application of the model, we assume that the entire enzy- on-times (A) and the velocity (Figure 3). Indeed, at low forces, matic cycle has only one rate-determining step. Relaxation of where the velocity is constant, we observe an exponential decay this assumption (i.e., if the rates of two or more substeps of constant, F ¼11.673.2 pN, nearly identical to that of the on-times. the cycle are comparable) would decrease the randomness of For higher forces, the decrease in velocity causes the measured mean distance to drop off even more quickly. Inset: A typical the distribution of times between steps (Svoboda et al, 1994); distribution of distances, and exponential fit; data taken from in turn, the estimated step size would necessarily increase to B300 events at F¼ 24 pN and 5 mM ATP. account for the measured level of noise. Thus, our estimate of 1272 bp is technically a lower bound. We see no significant variation in the step size with ATP concentrations of 1 or 2434 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al tentative hypothesis was that loop extrusion was due to the activity of two connected motors, with each translocating, Event signal but in opposite directions (Ip et al, 2003). Since FtsK is an AAAþ protein, it is probable that each motor is a hexamer encircling the DNA; thus two connected motors would form a double ring. We cannot rule out that each complex contains more than two motors; on the contrary, the characteristics of direction-reversal events are best explained by an FtsK 50C Fit to model complex containing three or more motors (see below). The formation of such higher order complexes is compatible with previously published electron microscopy and gel filtration Noise signal data on FtsK (Aussel et al, 2002). 50C A single motor is active during loop extrusion The range of measured timespans indicates that each event is due to only a single complex of FtsK . As mentioned, each 50C complex might be composed of several functional hexameric motors, two of which could bind to the DNA and form a loop. However, we argue that only one motor is translocating during the observed events: at low forces, the processivity increases to the extent that we see full-length, constant- Figure 5 Power spectrum analysis of the variance in bead height velocity events (in which the bead starts at full height and during (circles) and in the absence of (squares) protein activity. is brought smoothly to the capillary surface). If loops were Each set of points is the average of 26 segments of a data trace extruded by two motors simultaneously, a full-length, con- acquired at 5 mM ATP and F¼ 19 pN. The spectrum of noise during protein activity shows a clear 1/f increase over the spectrum of the stant-speed event would only occur if each motor bound measurement noise alone. Note that the latter spectrum is calcu- exactly to the middle of the DNA and worked in opposite lated from quiescent segments between the bursts of activity that directions until each hit a surface (the bead or the capillary). give rise to the former spectrum, and is thus a nearly simultaneous Such a precise starting position should be rare, yet we measure of the background noise. This increase is due to stochastic observe this type of event frequently at low force. Further variations of the motion of the protein, and is proportional to the protein’s step size d. The line is a fit to the predicted increase evidence disfavoring multiple active motors is the appearance 2 2 (Charvin et al, 2002): y¼ d/vS/2p f þ b, where /vS is the mean of direction-reversal events, in which the velocities of descent velocity and b accounts for the (white) measurement noise. From and ascent are nearly equivalent (Figure 2A). This process is many such fits, we find that, if the FtsK enzymatic cycle has a 50C difficult to explain if two motors are acting, since both motors single rate-determining step, it advances 1272 bp per cycle. would have to switch directions simultaneously. For these reasons, we conclude that during each measured event, only one motor is translocating. 5 mM or forces between 5 and 19 pN. If we assume the active motor is a hexamer (Aussel et al, 2002), and that 100% of the Localizing the protein to a DNA extremity added protein in the bulk ATPase measurement were active, Although only one motor is active, loop formation requires we can use the maximum velocities of translocation the FtsK complex to contact the DNA in two locations. In 50C (B6.7 kbp/s) and ATP hydrolysis (B30 ATP/monomer/s) to what manner could the complex form a second, nontranslo- estimate that the FtsK motor moves B37 bp per hydro- cating contact? The protein could possibly have a second 50C lyzed ATP. Given the step size, this leads to the impossible (immobile) type of DNA-binding domain, but this is unlikely result that less than one ATP is hydrolyzed per enzymatic for an aforementioned reason: full-length, constant-velocity cycle. It is important to note that this does not invalidate our events require that the protein begins translocation at one of result for the lower bound of the step size: a much more likely the extremities of the DNA, and random DNA binding would explanation is that a significant fraction of the protein in the not efficiently localize the protein to an extremity. Instead, we ATPase assay was inactive; this would not affect the single- suggest that the second binding location is also a motor molecule measurement. bound to the DNA, but stalled at the bead or capillary surface, as diagrammed in Figure 6. In this scenario, a diffusing complex first binds to the DNA through one motor, which Discussion translocates (without loop formation or a change in bead FtsK forms loops of DNA height) and transports the complex to a surface, where the 50C We attribute the observed transient decreases in DNA exten- motor stalls but remains bound to the DNA. Other motors in sion (Figure 1B) to the extrusion of loops of DNA by the complex are then free to bind; translocation of these translocating protein complexes: a protein solely moving motors causes loop formation and reduces the bead height. along a DNA molecule will cause no change in extension in The complex is localized to the extremity of the DNA, as our assay, and since each molecule is nicked we rule out required to explain the measured full-length events. The rare shortening due to an accumulation of supercoils. Formation events with twice the normal velocity (see Supplementary of DNA loops has been postulated before to explain the data) occur when a second motor binds before the first has topological modification of DNA substrates by FtsK stalled, resulting in a change in bead height at twice the 50C complexes (Aussel et al, 2002; Ip et al, 2003). A recurrent single-motor rate. Finally, this model explains the multiple &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2435 | | Single-molecule studies of FtsK translocation OA Saleh et al independent biochemical steps (involving no motion along the DNA, but rather, for example, ATP binding or a change in the protein’s internal conformation). The mechanical step requires the protein to change the height of the bead by a distance d and perform a work against the force of Fd; thus, the kinetic barrier of that step will increase at high hindering forces, decreasing the rate. If, at zero force, the mechanical step is much faster than the biochemical step, the zero-force velocity v will depend only on the step size and the bio- chemical step rate. As the force increases, the mechanical step rate slows until it becomes comparable to the biochem- ical step rate. For small F, the biochemical step determines the rate (and the velocity equals v , independent of the force), while for large F the mechanical step determines the rate (and the velocity decreases with force). This description qualita- Figure 6 Diagram of proposed steps in loop formation by FtsK tively matches our data at 5 mM ATP. Furthermore, it is 50C complexes. (A) Free complexes, containing several identical (prob- consistent with the 1 mM ATP data, where no velocity ably hexameric) motors, diffuse into the vicinity of the DNA. (B) decrease is seen up to F¼ 29 pN. The decrease to 1 mM ATP The complexes bind the DNA through a single motor, which slows the ATP-binding process, thus slowing the rate of the translocates and carries the complex toward an extremity of the DNA without changing the bead height. (C) Upon reaching the biochemical steps. To make the mechanical step rate compar- surface, the motor stalls but does not unbind, allowing other motors able to this lowered biochemical rate (i.e., to see a velocity in the complex to bind and translocate. (D) Translocation of the decrease) will thus require a larger force than was needed at second motor extrudes a loop of DNA, and decreases the bead 5 mM ATP; we apparently do not reach this regime at the height at the single-motor velocity. The decrease can be halted by (E) unbinding of either motor, leading to a fast recovery of the bead maximum force of 29 pN utilized in the 1 mM data. height, or (F) reversal of the translocation direction, and a slow For assisting forces, our data notably deviate from the recovery of bead height. We believe that direction reversal proceeds above model and the measurements on RNA polymerase through unbinding of the translocating motor, followed by binding (Neuman et al, 2003); in both, there is a continuation of and translocation by an oppositely directed motor from the same complex (see Discussion). the constant-velocity plateau. Instead, we observe a clear increase in velocity for large assisting forces (Figure 3). The velocity increase is not likely due to a change in the enzy- timescales in the off-time distributions (see Supplementary matic turnover rate (since it is unlikely that assisting force data): short off-times occur when only one of the two motors would quicken the limiting biochemical processes), but unbinds, leaving the complex in close proximity to the DNA rather due to an increase in the mechanical step size. and thus enabling fast rebinding, while long off-times occur Supporting this, we note that the velocity is still dependent when both motors unbind, and rebinding is limited by free on ATP in this regime; thus, the biochemical processes are diffusion of the protein complexes. still rate-determining. However, since our data are sparse at large negative forces due to the rarity of direction reversals Brief motor unbinding accompanies direction reversal (inset, Figure 2A) and the decrease in event size (Figure 4A), As shown in the inset to Figure 2A, direction reversal is less we cannot rule out the existence of a completely separate likely at higher forces. This can be explained by assuming mechanism of forward motion. that direction reversal first involves unbinding of the translo- cating motor, followed immediately by binding of an oppo- Force dependence of binding statistics indicates sitely directed motor: the observed decrease in binding rate a distortion of the DNA with force (i.e., the increase in off-time, see Figure 4A) would The mean value of the on-time decreases exponentially with then explain the decrease in the probability of direction force (see Figure 4A) until, at high hindering force, protein reversal with force (inset, Figure 2A). The oppositely directed activity is limited by this parameter. The exponential depen- motor could, in principle, be an inverted configuration of the dence can be accounted for by assuming that the DNA length first motor, but we consider it more likely that it is a different when bound by FtsK is shorter than when free. We model 50C (but nearby) motor within the same complex. this effect as a two-state system (Evans and Ritchie, 1997; Rief et al, 1998), where the bound and unbound states are Considerations on the mechanochemistry of the FtsK separated by a transition state with a higher free energy. Any 50C motor DNA length change l between the bound and transition states The force–velocity relationships of FtsK (Figure 3) show a would require unbinding to perform a work Fl against the 50C clear plateau for small forces of either orientation, along with applied force. This would affect the mean on-time (the a decrease in velocity (at saturating 5 mM ATP) for large inverse of the mean unbinding rate) by a factor exp(Fl/ hindering forces. This behavior is qualitatively similar to both kT), consistent with our observations of an exponential theoretical predictions (Keller and Bustamante, 2000) and dependence on force. Based on the fitted exponential decay previous results obtained on RNA polymerase (Wang et al, constant of 11.370.9 pN, we estimate that unbinding of 1998; Neuman et al, 2003). The behavior is indicative of two FtsK causes the DNA length to increase by 50C types of sequential steps in the reaction that causes forward lX0.3670.03 nm (the estimate is a lower bound since the movement of the protein: a force-dependent mechanical step measurement is not sensitive to any further change in DNA (involving motion along the DNA), and one or more force- length between the transition and unbound state; see 2436 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al Supplementary data). This length change is much smaller activities of FtsK and these other proteins will create a tension than the minimal 80 bp binding site required to stimulate in the DNA analogous to the force we apply to the bead in our FtsK ’s ATPase activity (Massey et al, 2004). Therefore, it is assay. Just as we observe (Figure 4B), this tension will 50C consistent only with a small bend in the DNA induced at the decrease the distance FtsK can travel before unbinding. protein’s binding site. FtsK motors that pump in the wrong direction will thus quickly fall off, allowing other, perhaps correctly oriented, Implications of direction reversal for models of FtsK motors to bind. Although the lack of structural data on FtsK polarity precludes affirmation, the formation of higher order com- It has been suggested that some chromosomal oligomeric plexes by FtsK and the resulting in vitro direction-reversal 50C sequences, with a high skew that inverts at dif, could direct activity might indicate that higher order complexes are also formed in vivo by FtsK, which would facilitate this process. the FtsK translocation process (Perals et al, 2000; Corre and Motors pumping in the correct direction will not create Louarn, 2002), just like Chi sequences alter the enzymatic properties of RecBCD complexes (Spies et al, 2003). In this tension in the DNA, and will translocate a correspondingly regard, direction reversal is surprising because it indicates greater distance. In this way, FtsK would clear any misplaced that FtsK can move in both directions on the same segment chromosomal loops that pass through the septum. 50C of DNA. Furthermore, we observe full-length direction-rever- Interestingly, this model could be applied to sporulation in B. subtilis, assuming that condensation of DNA in the pre- sal translocation events on a lambda DNA fragment of spore generates sufficient tension. approximately half the size of the phage genome; introduc- tion of a complete phage lambda genome has been shown to This force-rectified translocation model applies to segrega- perturb chromosome polarity in vivo (Corre et al, 2000). To tion of normal daughter chromosomes and to resolution of confirm this finding, we perform experiments on a fragment chromosome dimers. For normal chromosome segregation, of the E. coli genome that was shown to be polarized (Perals FtsK would simply accelerate the clearing process; both et al, 2000). Full-length translocation events after direction genetic and cellular biology data indicate that FtsK is not absolutely required for cohesion and mid-cell positioning of reversal are again observed (Figure 2B). We conclude that the oligomeric DNA motifs that polarize the E. coli and lambda the large terminus region of the E. coli chromosome (Capiaux genomes do not constitute absolute blocks to DNA transloca- et al, 2002; Corre and Louarn, 2002). In the presence of tion by FtsK . We cannot rule out the possibilities that their chromosome dimers, misplaced loops could still occur; FtsK 50C action is probabilistic, or that it requires the presence of the would clear these loops from the septum until there remained N-terminal or linker domains of FtsK. However, our data raise only the two chromosome-connecting strands that contain the dif sites required for XerC/D recombination. the possibility that DNA sequence information plays only an indirect role on polarity of translocation by FtsK. For exam- ple, an additional protein could bind to specific DNA se- FtsK ’s high velocity enables fast processing quences and block FtsK translocation, just like Tus binding to 50C of misaligned DNA Ter sites can stop replication forks (Kamada et al, 1996), or, FtsK ’s maximum velocity of B6.7 kbp/s makes it the rather than acting on FtsK, the DNA sequence could direct 50C fastest DNA-based motor protein yet measured. Speeds simi- progressive, oriented condensation of the nucleoids, thereby lar to our measurements have been estimated for the total imposing that the dif region is the last chromosomal section transfer rate of the B. subtilis genome into a nascent spore by to be moved by FtsK. SpoIIIE (Errington et al, 2001). As mentioned above, we do not expect FtsK to mobilize such a large fraction of the E. coli A force-rectified translocation model could impose genome, but rather a tangle of DNA loops. Unravelling such directionality a tangle could require loops to be successively processed Based on the observed sensitivity of the travelled distance to several times, as has been reported in vitro for replicative force (Figure 4B), we propose an alternate mechanism that catenanes (Ip et al, 2003); thus FtsK ’s high speed could be could account for in vivo FtsK directionality. The parameters 50C needed to complete multiple processing of misaligned loops of the exponential curve fit of mean on-time versus force quickly. (Figure 4A), along with the maximum speed, indicate that at zero force the FtsK complex will travel, on average, 50C B11 kbp per binding event. Since DNA translocation most likely involves the binding of two identical motor units Materials and methods (Figure 6), the zero-force mean distance for each motor is Protein, substrate DNA, and slide preparation then twice that of the complex, orB22 kbp. This is much less FtsK is purified as described in Aussel et al (2002). l DNA (bp 50C than the total length of the E. coli chromosome (4.5 Mbp). 25 882–45 679) is amplified by long-range PCR. E. coli chromosomal However, the chromosome is compacted in the cell and it is DNA (bp 8877–22 422 after the XerC-binding site of dif) is purified from plasmid pFC94 (Perals et al, 2000). DNA substrates are ligated highly probable that to clear the septum, FtsK works on DNA to biotin- and digoxigenin-modified DNA fragments, which are loops of sizes similar to the zero-force mean distance. prepared by PCR using cognate modified nucleotides. The resulting We propose that a septum-bound FtsK motor, upon bind- constructs are incubated with 4.5 mm diameter paramagnetic ing a chromosomal loop, can translocate in either direction. streptavidin beads and added to 1-mm square cross-section glass However, if pumping in the wrong direction, it will pull capillaries, which had been washed with 0.1 M NaOH, coated with SigmaCote, and sequentially incubated with solutions of anti- against the large fraction of condensed DNA and work against digoxigenin and bovine serum albumin (BSA). We insure that the other proteins in the nascent daughter cell that maintain the beads we use are bound to the capillary by a single, nicked DNA chromosome position, such as MukB and active next-genera- molecule, and not a single unnicked, molecule or multiple tion DNA replisomes (Sherratt, 2003). The counteracting molecules, as described in Strick et al (1996, 1998). &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2437 | | Single-molecule studies of FtsK translocation OA Saleh et al Measurement of applied force and bead height slow recoveries (inset, Figure 2A), the number of slow recovery segments of a certain minimum size (typically 0.5 mm; less at higher The image of each bead is captured by a CCD camera at 60 Hz and processed, using custom-written software, to give the 3D bead forces) are counted, and divided by the total number of events of position. Before protein addition, we calibrate the apparatus for that size; the threshold is needed due to the difficulty in each bead by measuring the applied force as a function of magnet/ characterizing very short recoveries. For the fluctuation analysis, capillary separation distance. The applied force is measured by within a data set, only long (40.75 mm) events are analyzed. A monitoring the mean squared transverse fluctuations /dx S of the straight line is fit to, and subtracted from, each event to acquire the bead (see Figure 1A), and applying the equipartition theorem: difference at each time point. The mean variance from the fit line is 1 1 2 kT ¼ hdx iF=L, where L is the measured bead height (Strick et al, computed for each event; typically, the mean variance across all 2 2 1996). Microscope drift is removed by simultaneously tracking a events forms a compact distribution. Outliers from this distribution reference bead stuck to the capillary surface, and subtracting its (more than 2 standard deviations larger than the mean) sometimes height from that of the experimental bead. occur, and are considered to contain anomalous noise; thus, the corresponding events are discarded. Power spectra are then Addition of protein to the capillary calculated from the suitable events using standard algorithms. Experiments are performed in a buffer containing 10 mM MgCl , 10 mM Tris pH 7.9, 50 mM NaCl, 1 mM DTT, 0.01% Triton X-100, Bulk ATPase assay and ATP. In all, 5–10 ml of 200 nM FtsK monomers is added to 50C Reactions are performed with 100 nM of FtsK (monomer), 5 nM 50C 200 ml of buffer in the capillary, and gently mixed. of 3 kb supercoiled plasmid DNA, 1 nM [a P]ATP and cold ATP in 10 ml of 10 mM Tris–HCl, pH 7.9, 10 mM MgCl , 50 mM NaCl, and Long data acquisitions 1 mM DTT. Reactions are incubated for 3 min before being stopped Activity resulting from a single addition of protein can last for with EDTA and excess ATP. The ratio of ATP to ADP is analyzed by hours. To control for possible degradation of the protein or ATP in thin layer chromatography. We check that our measurements that time, we measure the activity at a reference force multiple correspond to initial rates and that DNA is in a 10-fold excess in times throughout the experiment. We never observe a decrease in the reaction (data not shown). velocity in those reference measurements. In some long measure- ments, we do observe an increase in the mean off-time; we attribute this to a decrease in the active protein concentration through Supplementary data degradation or nonspecific adsorption to the capillary. Data sets Supplementary data are available at The EMBO Journal Online. containing such a decrease are not used to construct the off-time plot in Figure 4A. Acknowledgements Data analysis Measured traces of bead height correspond directly to DNA We thank D Bensimon, V Croquette, J-M Louarn, and F Cornet for extension; no filtering has been applied to any of the plots shown. constant support and helpful discussions. We thank V Croquette for Translocation velocities, lengths, and on- and off-times are sharing analysis software, and J-Y Bouet, G Charvin, K Neuman, extracted by analyzing each data set with custom-written software and T Lionnet for critical reading of the manuscript. Research was that applies a filter (Chung and Kennedy, 1991) in order to identify funded by the CNRS and the Ecole Normale Supe´rieure. Research in events automatically, while still measuring each parameter from the Paris was supported by grants from the French Research Ministry unfiltered data. Data quoted in units of base pairs have been ACI Jeune Chercheur program and from the EU MolSwitch program. corrected for the difference between measured DNA extension and Research in Toulouse was supported by grants from the CNRS ATIP the true contour length by applying the worm-like chain model for program and from the French Research Ministry Fundamental DNA elasticity (Bouchiat et al, 1999). To calculate the fraction of Microbiology ACI program. References Aussel L, Barre FX, Aroyo M, Stasiak A, Stasiak AZ, Sherratt D Corre J, Patte J, Louarn JM (2000) Prophage lambda induces (2002) FtsK is a DNA motor protein that activates chromosome terminal recombination in Escherichia coli by inhibiting dimer resolution by switching the catalytic state of the XerC and chromosome dimer resolution. An orientation-dependent cis-ef- XerD recombinases. Cell 108: 195–205 fect lending support to bipolarization of the terminus. Genetics Bath J, Wu LJ, Errington J, Wang JC (2000) Role of Bacillus subtilis 154: 39–48 SpoIIIE in DNA transport across the mother cell-prespore division Donachie WD (2002) FtsK: Maxwell’s demon? Mol Cell 9: 206–207 septum. Science 290: 995–997 Draper GC, McLennan N, Begg K, Masters M, Donachie WD (1998) Begg KJ, Dewar SJ, Donachie WD (1995) A new Escherichia coli cell Only the N-terminal domain of FtsK functions in cell division. division gene, ftsK. 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EMBO Rep 3: 532–536 J 22: 6399–6407 Charvin G, Bensimon D, Croquette V (2002) On the relation Kamada K, Horiuchi T, Ohsumi K, Shimamoto N, Morikawa K between noise spectra and the distribution of time between (1996) Structure of a replication-terminator protein complexed steps for single molecular motors. Single Molecules 3: 43–48 with DNA. Nature 383: 598–603 Chen JC, Beckwith J (2001) FtsQ, FtsL and FtsI require FtsK, but not Keller D, Bustamante C (2000) The mechanochemistry of molecular FtsN, for co-localization with FtsZ during Escherichia coli cell motors. Biophys J 78: 541–556 division. Mol Microbiol 42: 395–413 Lau IF, Filipe SR, Soballe B, Okstad OA, Barre FX, Sherratt DJ (2003) Chung SH, Kennedy RA (1991) Forward-backward nonlinear filter- Spatial and temporal organization of replicating Escherichia coli ing technique for extracting small biological signals from noise. chromosomes. 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Mol Microbiol Sharp MD, Pogliano K (2002) Rote of cell-specific SpoIIIE assembly 49: 241–249 in polarity of DNA transfer. Science 295: 137–139 Yu XC, Tran AH, Sun Q, Margolin W (1998a) Localization of cell Sherratt DJ (2003) Bacterial chromosome dynamics. Science 301: division protein FtsK to the Escherichia coli septum and identifi- 780–785 cation of a potential N-terminal targeting domain. J Bacteriol 180: Spies M, Bianco PR, Dillingham MS, Handa N, Baskin RJ, 1296–1304 Kowalczykowski SC (2003) A molecular throttle: the recombina- Yu XC, Weihe EK, Margolin W (1998b) Role of the C terminus of tion hotspot chi controls DNA translocation by the RecBCD heli- FtsK in Escherichia coli chromosome segregation. J Bacteriol 180: case. Cell 114: 647–654 6424–6428 &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2439 | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Fast, DNA‐sequence independent translocation by FtsK in a single‐molecule experiment

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10.1038/sj.emboj.7600242
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Abstract

The EMBO Journal (2004) 23, 2430–2439 & 2004 European Molecular Biology Organization All Rights Reserved 0261-4189/04 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Fast, DNA-sequence independent translocation by FtsK in a single-molecule experiment 1 2 such, they are thought to form hexameric motors, which are Omar A Saleh , Corine Pe´rals , 2, responsible for the active translocation process. They are Franc¸ois-Xavier Barre * and 1,3, involved in a wide range of functions: TraSA is encoded by Jean-Franc¸ois Allemand * the plasmid pSAM2 of Streptomyces ambofaciens,and is Laboratoire de Physique Statistique et Departement de Biologie, Ecole 2 responsible for the transfer of the plasmid from donor cells Normale Superieure, Paris, France, Laboratoire de Microbiologie et into acceptor cells during conjugation (Possoz et al, 2001). ´ ´ ´ de Genetique Moleculaire, Toulouse, France and Laboratoire Pasteur, ´ ´ Departement de Chimie, Ecole Normale Superieure, Paris, France SpoIIIE is responsible for the transport of a chromosome into the small polar prespore compartment during sporulation in Escherichia coli FtsK is an essential cell division protein, Bacillus subtilis (Bath et al, 2000). FtsK is an essential which is thought to pump chromosomal DNA through the Escherichia coli cell division protein (Begg et al, 1995). Its closing septum in an oriented manner by following DNA N-terminal membrane domain is localized to the division sequence polarity. Here, we perform single-molecule mea- septum (Wang and Lutkenhaus, 1998; Yu et al, 1998a), and is surements of translocation by FtsK , a derivative that 50C necessary for its formation (Draper et al, 1998; Chen and functions as a DNA translocase in vitro. FtsK transloca- Beckwith, 2001). Its C-terminal motor domain (FtsK )is 50C tion follows Michaelis–Menten kinetics, with a maximum implicated in chromosome segregation (Liu et al, 1998; Yu speed of B6.7 kbp/s. We present results on the effect of et al, 1998b). Indeed, FtsK has two essential roles in applied force on the speed, distance translocated, and the chromosome dimer resolution (see Aussel et al, 2002; mean times during and between protein activity. Capiaux et al, 2002; Corre and Louarn, 2002, and references Surprisingly, we observe that FtsK can spontaneously 50C therein for review). Chromosome dimers, formed by homo- reverse its translocation direction on a fragment of E. coli logous recombination in organisms with circular chromo- chromosomal DNA, indicating that DNA sequence is not somes, are a threat to normal repartitioning of genetic the sole determinant of translocation direction. We con- information at cell division. In E. coli, chromosome dimers clude that in vivo polarization of FtsK translocation could are resolved into monomers by the addition of a crossover at require the presence of cofactors; alternatively, we propose a specific 28 bp site, dif, by two tyrosine recombinases, XerC a model in which tension in the DNA directs FtsK translo- and XerD. Chromosome dimer resolution first requires colo- cation. calization of the dif sites; this is accomplished by transloca- The EMBO Journal (2004) 23, 2430–2439. doi:10.1038/ tion of the chromosomes by FtsK (Capiaux et al, 2002; Corre sj.emboj.7600242; Published online 27 May 2004 and Louarn, 2002). Secondly, the recombination reaction Subject Categories: genome stability & dynamics; itself is activated by FtsK (Aussel et al, 2002), probably microbiology & pathogens through a direct interaction with XerC/D (Yates et al, 2003; Keywords: chromosome segregation; DNA translocation; Massey et al, 2004). In addition, DNA translocation by FtsK FtsK; magnetic tweezers; molecular motor could participate in segregation of normal chromosomes (Yu et al, 1998b; Donachie, 2002; Lau et al, 2003). Coupled to these processes, there must be a mechanism of translocation polarity; that is, an orienting mechanism that Introduction ensures the DNA is transported in the correct direction. In B. subtilis, it has been argued that preferential assembly of Active transportation of DNA from one cellular compartment SpoIIIE in one daughter cell establishes polarity in chromo- to another is central to many biological processes, such as some partitioning during sporulation (Sharp and Pogliano, viral DNA packaging, conjugation, sporulation, and cell divi- 2002). In normal vegetative growth in E. coli, in contrast to sion. In bacteria, a new class of proteins was recently sporulating B. subtilis, there is no obvious morphological nor identified that mediate the transport of double-stranded protein expression difference between the two daughter cells DNA through cell membranes and cell walls: the FtsK/ that could explain asymmetric assembly of FtsK. Instead, it SpoIIIE/TraSA family (Bath et al, 2000; Possoz et al, 2001; has been postulated that FtsK translocation polarity is defined Aussel et al, 2002). These proteins belong to the AAAþ by chromosomal sequence polarity, that is, a high skew of (ATPase Associated with various Activities) superfamily. As oligomeric sequences along the two replichores that inverts at *Corresponding authors. Franc¸ois-Xavier Barre, Laboratoire de dif (Salzberg et al, 1998; Lobry and Louarn, 2003). Microbiologie et de Ge´ne´tique Mole´culaire, 118 Route de Narbonne, Chromosome dimer resolution is effective only when dif is 31062 Toulouse, France. Tel.: þ 33 5 61 33 59 86; located within a narrow zone of the chromosome (Cornet Fax: þ 33 5 61 33 58 86; E-mail: [email protected] or Jean-Franc¸ois et al, 1996), functionally defined as the junction between Allemand, Laboratoire de Physique Statistique, Ecole Normale Supe´rieure, 24, Rue Lhomond, 75005 Paris, France. DNA segments of opposite sequence polarity (Corre et al, Tel.: þ33 144323496; Fax: þ33 144323433; 2000; Perals et al, 2000). Sequence polarity can be perturbed E-mail: [email protected] by the introduction, close to dif, of exogenous DNA, such as the genome of phage lambda (Corre et al, 2000). This change Received: 23 February 2004; accepted: 27 April 2004; published online: 27 May 2004 inactivates chromosome dimer resolution, and augments the 2430 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al probability of endogenous recombination in an FtsK-depen- Results dent manner (Corre and Louarn, 2002). This has led to the FtsK causes transient decreases of DNA extension 50C idea that polarization of the chromosome sequence could Magnetic beads (4.5 mm) are tethered to a glass capillary by a direct FtsK’s translocation so as to juxtapose the chromoso- section of DNA (see Figure 1A), as described in Materials and mal dif sites; this mechanism could also assist in clearing the methods. We use a portion of lambda DNA in all experiments septum of any residual DNA. presented, with one exception (noted below) in which we use In vitro evidence for DNA translocation by the ATPase- motor domains of SpoIIIE and FtsK has been obtained, but all the assays employed were indirect, precluding detailed ana- lysis of the translocation reaction (Bath et al, 2000; Aussel et al, 2002; Ip et al, 2003). Here, we directly monitor the DNA translocase activity of FtsK , an active, oligomeric C-term- 50C inal truncation of FtsK (Aussel et al, 2002), at the single- molecule level using magnetic tweezers (Strick et al, 1996). We measure, in various conditions of applied force and ATP concentration, the velocity, length, and duration of individual translocation events. Using these data, we deduce basic mechanochemical parameters of the protein’s translocation reaction. Surprisingly, we find that FtsK can spontaneously re- 50C verse its translocation direction, and thus travel both ways on the same segment of DNA. We test translocation on both lambda genomic DNA and an E. coli chromosomal fragment that has been shown to be polarized in vivo (Perals et al, 2000). No bias in translocation is observed on either of these substrates, suggesting that DNA sequence polarity does not directly affect FtsK activity. We conclude that in vivo 50C polarization of FtsK translocation could require additional cofactors, such as domains of the full protein not included in the truncation used here. Alternatively, we propose a me- chanism in which force, rather than DNA sequence, directs the protein’s in vivo motion. Figure 1 (A) Schematic representation of the measurement appa- ratus. A magnetic bead is tethered to a glass capillary by a single, nicked DNA molecule. Magnets above the capillary create a field gradient that pulls on the bead with a force F, which is determined by measuring the bead’s lateral fluctuations dx. By optically track- ing the bead, we measure the extension L of the DNA with time. (B) Typical measurement of DNA extension, both with and without FtsK in the solution. The plotted data were measured at 50C F¼ 10.7 pN and with 5 mM ATP; the data sets have been offset for clarity. We attribute the transient decreases in DNA extension to the creation of loops of DNA by single complexes of FtsK .(C)A 50C typical individual event extracted from the data shown in (B). All events begin with a constant-velocity decrease in DNA extension; we attribute the slope of the descent to the translocation velocity of the complex. The velocity, distance travelled d, and on-time are easily measured because of the well-defined event shape. As sketched, loop formation requires the complex to bind the DNA in two locations; see Figure 6 and Discussion for details. (D) FtsK 50C activity versus ATP concentration for both bulk (rate of ATP consumption; red curve) and single molecule (translocation velo- city; green points) assays. Both data sets are well fit by the Michaelis–Menten equation V [ATP]/([ATP]þ K ), with, respec- max m tively, V ¼ 3072 ATP/s and 6.770.1 kbp/s, and K ¼ 270740 max m and 330720 mM. The fit to the single-molecule data (blue curve) is shown; it is highly coincident with the bulk data fit, which is omitted for clarity. Single-molecule data are taken at FB5 pN, and each point is the average of typically 100 events; plotted bars indicate standard error. Inset: Histogram of measured translocation velocities (open circles) with best-fit Gaussian curve (solid line); data taken at 5 mM ATP and F¼ 6 pN. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2431 | | Single-molecule studies of FtsK translocation OA Saleh et al a fragment of E. coli chromosomal DNA. We search for beads presence of DNA (Aussel et al, 2002), are consistent with a that are tethered by a single, nicked DNA molecule; while we mechanism in which ATP consumption is restricted to DNA- are able to identify and utilize unnicked DNA molecules, we bound FtsK , and leads directly to DNA translocation. 50C concentrate here on nicked molecules so as to avoid the topological complications of supercoiling. Once a suitable Reversal of the translocation direction bead is found, we flush the capillary with ATP-laden buffer, After the initial translocation, the recovery back to the add purified FtsK monomers to a final concentration of original bead height occurs in one of two ways, as shown 50C 5–10 nM, and begin tracking the DNA extension with time. in Figure 2A. The first type of recovery is an abrupt jump that After a short time, many transient decreases in DNA exten- occurs at speeds 420 mm/s, which is too fast to be resolved sion are observed, as shown in Figure 1B. Preliminary data on in our system. This is consistent with the protein completely unnicked molecules (OA Saleh, S Bigot, FX Barre and JF unbinding, and the bead rising at a rate determined by the Allemand, in preparation) are in general very similar to applied force and hydrodynamic Stokes drag (e.g. a 2 pN nicked-molecule data; thus, nicks appear not to affect the force will move a 4.5 mm diameter bead through water at a protein activity. The typical waiting time between events terminal velocity of B50 mm/s). The second type of recovery (B5 s) is greater than the typical event timespan (B0.2 s), is a slower, constant-velocity increase, with a slope (at low indicating that the measured activity is due to the action of a forces) nearly equivalent to that of the decrease. We believe single protein complex. Note that the structure of FtsK in that slow recoveries are caused by a single complex that, after 50C solution is unknown, and apparently multimeric (Aussel et al, decreasing the bead height, reverses its translocation direc- 2002); throughout the text, we refer to the active protein unit tion and causes the bead to rise (see Discussion and Figure 2A as a complex. The events continue to occur for a long time sketches). Both types of recoveries occur at all forces; at a without need of additional proteins or ATP. We detect no given force, events with slow and fast recoveries are ran- events in the absence of either ATP or FtsK , and we find domly interspersed. However, it is clear that the frequency of 50C that existing activity stops upon addition of EDTA to a final occurrence of direction reversals depends on the applied concentration of 20 mM. From these observations, we con- force: the proportion of slow recoveries decreases from clude that the events are caused by the ATP-dependent DNA B50% at low forces to nearly zero at high forces (inset, translocation activity of FtsK . Figure 2A). Rarely, we observe events with more complicated 50C mixed recoveries, wherein the bead height first increases slowly, then abruptly, or vice versa (see Supplementary data). Translocation velocity represents protein activity Direction reversal has potential implications for the in vivo Each event begins with a linear decrease in bead height (Figure 1C), from which we can extract both the translocation mechanism of FtsK translocation polarity, as it shows that, on velocity and the total distance travelled by the protein. Very the section of lambda DNA utilized, the protein can travel in rarely, we observe events containing pauses that interrupt the both directions on the same DNA sequence. To further constant-velocity decrease. The pauses have no correlation explore this effect, we perform measurements on a fragment of E. coli chromosomal DNA with a proven in vivo polarity with position along the DNA, and only occur in situations effect (Perals et al, 2000). As shown in Figure 2B, we still where many protein complexes appear to be active. We attribute pausing to protein–protein interactions, and do not observe direction reversal events on the E. coli DNA, with no characterize them here; see Supplementary data for more difference in either the shape of individual events, or the information. Within a single measurement (i.e., on one DNA frequency of occurrence of slow recoveries. Furthermore, full- molecule, and at constant force and ATP concentration), the length direction reversal events occur (wherein the bead is translocation velocity measured from all events is normally brought from full height down to the capillary surface, then back up again, all at constant speed; see Figure 2B), indicat- distributed (inset, Figure 1D). Infrequently, an event is ob- ing that no subsection of the E. coli DNA fragment has a served with almost precisely double the mean translocation velocity of other events in the data set (see Supplementary definitive effect on the translocation direction of the complex. data). Such events presumably arise from two-motor activity (see Discussion), and are removed from all the analysis Force dependence of the translocation velocity presented. The width of the distribution is accounted for by In our assay, the applied force F depends only on the distance measurement noise and variations due to the stochastic of the magnets to the capillary; we can easily adjust this stepping motion of the motor protein (Svoboda et al, 1994); distance to study the dependence of translocation velocity on thus, within the experimental noise, we observe no protein- force. During initial translocation, the protein lowers the to-protein or sequence-based velocity variation. bead, and must perform work against the applied force. In To confirm that FtsK ’s ATPase activity causes its trans- contrast, during direction-reversed translocation, the bead 50C location, we compare, at various ATP concentrations, the rises, so the protein works with the (upward) force. single-molecule velocity with the bulk ATPase activity mea- Thus, we can use initial translocation to measure the sured in an excess of DNA (insuring each functional protein is velocity with forces hindering the protein’s motion, and active). As shown in Figure 1D, the assays agree quite well: reversed translocation to measure the velocity with each independently indicates that FtsK follows simple forces assisting the motion (following previous practice, we 50C Michaelis–Menten kinetics with K B0.3 mM ATP and respec- define a hindering force as positive). In Figure 3, we show tive maximum velocities of B6.7 kbp/s and B30 ATP/ the force–velocity relationships measured in the presence of 1 FtsK monomer/s. Note that the latter is only a relative and 5 mM ATP. At both concentrations, there is a clear 50C measure since it ignores the presence of nonfunctional (mis- plateau of constant velocity for small forces, along with a folded) proteins. These results, in the light of previous data transition to increasingly higher velocities at large assisting showing that ATPase activity is highly stimulated in the forces; this transition occurs, respectively, at 4 and 11 pN 2432 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al F < 0 F > 0 Assisting Hindering force force 5 mM ATP 1 mM ATP Figure 3 Force–velocity relations for FtsK measured at 1 and 50C 5 mM ATP. At both ATP concentrations, there is a plateau of constant velocity for small forces. The velocity measured at 5 mM ATP begins to decrease above a hindering force of 15 pN, while no decrease is seen in the 1 mM data up to 29 pN. The hindering-force data indicate that the FtsK reaction pathway has force-indepen- 50C dent biochemical step(s) in series with force-dependent mechanical step(s) (see Discussion). Plotted bars indicate standard error; large error estimates indicate regimes where the event frequency and size are small (see Figure 4). Mean event length and on-time decrease with force We are unable to measure the velocity above B35 pN because of a strong decrease in the size of each event, and in the frequency of event occurrence. The length and duration (i.e., on-time) of each event correspond, respectively, to the distance travelled by the protein, and the time spent bound to the DNA, before unbinding or reversing direction (see Figure 1C). Within a measurement at a given force and ATP concentration, both distance and on-time vary from event to event. We find that the distribution of each is always exponential (see inset, Figure 2 (A) Illustration of different types of recoveries. After Figure 4B), indicating a constant unbinding probability decreasing, the DNA extension can recover its full length either with time (and thus distance, given the nearly constant abruptly (black curve) or slowly (red curve). As sketched, we velocity within a given measurement; see inset, Figure 1D); attribute the former to protein unbinding, and the latter to a reversal of the translocation direction. The fraction of events exhibiting a we can then fit an exponential curve to each distribution and slow recovery is force dependent, as plotted in the inset. The extract the mean distance and on-time for the given condi- distance travelled before a reversal of direction is random, and tions. In this way, we measure, at constant 5 mM ATP, the consistent with the distribution of distances travelled before un- variation of mean distance and on-time with applied force. As binding (see Figure 4). The events plotted were measured at 18 pN and 5 mM ATP, and are offset for clarity. The inset includes data shown in Figure 4A, the mean on-time decreases strongly, and taken at ATP concentrations from 0.5 to 5 mM; we observe no exponentially, with force. Fitting the data to t exp(F/F ) 0 0 variation in the slow fraction with ATP. (B) Translocation of a gives a decay constant F ¼11.370.9 pN and a mean on-time fragment of E. coli chromosomal DNA that has been shown to at zero force of t ¼1.670.3 s. The mean distance travelled have in vivo polar activity (Perals et al, 2000); data taken at 5 mM ATP and F¼ 1 pN. All the three plotted events have slow, direction- also decreases strongly with force, as shown in Figure 4B. reversal recoveries. In particular, in the first event, FtsK translo- 50C At low forces, the translocation velocity is fairly constant cates the entire length of the DNA in both directions, indicating that (Figure 3), so we expect the mean distance to vary in the no portion of the DNA sequence has a strong effect in biasing the same manner as the mean on-time. This is indeed the case: translocation direction. an exponential fit to the low-force points gives a decay constant F ¼11.673.2 pN, nearly equivalent to the decay for 1 and 5 mM ATP. At 5 mM ATP, the velocity begins to constant of the mean on-time. Above 20 pN, the mean clearly decrease for F415 pN; no such transition is seen in distance drops off more quickly due to the decrease in the 1 mM data up to F ¼ 29 pN. velocity at high forces. &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2433 | | / Single-molecule studies of FtsK translocation OA Saleh et al Force dependence of the off-times Along with the distances and on-times, we extract from each measurement distributions of the off-times, that is, the time duration between events (see Figure 1B). The off-time dis- tributions from a given measurement clearly do not follow a single exponential curve, but rather a curve that is the sum of (at least) two exponentials with, respectively, fast and slow time constants (see Supplementary data). The presence of Off-times fast and slow rebinding rates is presumably due to the varying timescales of processes that contribute to protein– DNA binding (see Discussion). We find strong variations in On-times the mean off-time for different measurements, which we F = 11.3 ± 0.9 pN attribute to variations in the amount and active percentage of the added protein. However, since a single addition of protein to the capillary results in long-lasting activity, we can vary the force and confidently measure the response of the off-times at constant protein concentration. As shown in Figure 4A, we find that the mean values of the off-time increase with force, indicating that binding rate of the protein to the DNA is decreased by the application of higher forces. Noise analysis reveals FtsK step size 50C The completion of an enzymatic cycle results in a single forward step of a motor protein. If the step size is larger than Exponential fit, the measurement noise, and the average time to complete an F = 11.6 ± 3.2 pN enzymatic cycle is larger than the time resolution of the measurement, it would be possible to observe individual steps directly. This is not the case here: we observe no discrete steps in the traces of bead position versus time. However, it is still possible to estimate the step size by considering the fluctuations in the measurement. In the absence of protein activity, the resolution of our measure- ment is limited by the bead’s Brownian fluctuations, which, at low frequency f, create a frequency-independent ‘white’ noise. During protein activity (i.e., during the linear decrease in bead height), we find that the measurement noise in- creases at low frequencies as 1/f , and thus cannot be attributed only to Brownian fluctuations (see Figure 5). Such a low-frequency increase in noise has been observed in previous single-molecule measurements (Svoboda et al, 1994), and has been shown to be proportional to the enzy- matic step size (Svoboda et al, 1994; Charvin et al, 2002); it is Figure 4 (A) Force dependence of the on- and off-times at 5 mM caused by the random distribution of the times between ATP. As with the distance travelled (see inset, B), the distribution of individual steps (Svoboda et al, 1994). on-times from many events within a measurement is exponential To estimate the step size, we select the active segment of a (data not shown), indicating a constant unbinding probability with time. We extract a mean on-time from each distribution through suitable event and compute, at each time point, the difference fitting an exponential function. Here, we plot the variation with between the bead’s measured position and its mean position force of the mean on-time (circles), and fit it with an exponential (as predicted by the mean velocity). We then find the power function t exp(F/F ). The mean on-time decreases with force with 0 0 spectrum of that difference, average the spectra over many a decay constant F ¼11.370.9 pN and an on-time at zero force of t ¼1.670.3 s. This exponential force dependence can be simply 0 events from a single measurement, and fit this average with explained if we assume that binding of the protein causes a the theoretically predicted curve (Charvin et al, 2002); an distortion that decreases the DNA length by at least example is shown in Figure 5. By performing this analysis, we 0.3670.03 nm (see Discussion). (B) Dependence of the mean estimate that the step size of FtsK is 1272 bp. In our distance travelled per event on force at 5 mM ATP. The mean 50C distance travelled at a given force should follow directly from the application of the model, we assume that the entire enzy- on-times (A) and the velocity (Figure 3). Indeed, at low forces, matic cycle has only one rate-determining step. Relaxation of where the velocity is constant, we observe an exponential decay this assumption (i.e., if the rates of two or more substeps of constant, F ¼11.673.2 pN, nearly identical to that of the on-times. the cycle are comparable) would decrease the randomness of For higher forces, the decrease in velocity causes the measured mean distance to drop off even more quickly. Inset: A typical the distribution of times between steps (Svoboda et al, 1994); distribution of distances, and exponential fit; data taken from in turn, the estimated step size would necessarily increase to B300 events at F¼ 24 pN and 5 mM ATP. account for the measured level of noise. Thus, our estimate of 1272 bp is technically a lower bound. We see no significant variation in the step size with ATP concentrations of 1 or 2434 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al tentative hypothesis was that loop extrusion was due to the activity of two connected motors, with each translocating, Event signal but in opposite directions (Ip et al, 2003). Since FtsK is an AAAþ protein, it is probable that each motor is a hexamer encircling the DNA; thus two connected motors would form a double ring. We cannot rule out that each complex contains more than two motors; on the contrary, the characteristics of direction-reversal events are best explained by an FtsK 50C Fit to model complex containing three or more motors (see below). The formation of such higher order complexes is compatible with previously published electron microscopy and gel filtration Noise signal data on FtsK (Aussel et al, 2002). 50C A single motor is active during loop extrusion The range of measured timespans indicates that each event is due to only a single complex of FtsK . As mentioned, each 50C complex might be composed of several functional hexameric motors, two of which could bind to the DNA and form a loop. However, we argue that only one motor is translocating during the observed events: at low forces, the processivity increases to the extent that we see full-length, constant- Figure 5 Power spectrum analysis of the variance in bead height velocity events (in which the bead starts at full height and during (circles) and in the absence of (squares) protein activity. is brought smoothly to the capillary surface). If loops were Each set of points is the average of 26 segments of a data trace extruded by two motors simultaneously, a full-length, con- acquired at 5 mM ATP and F¼ 19 pN. The spectrum of noise during protein activity shows a clear 1/f increase over the spectrum of the stant-speed event would only occur if each motor bound measurement noise alone. Note that the latter spectrum is calcu- exactly to the middle of the DNA and worked in opposite lated from quiescent segments between the bursts of activity that directions until each hit a surface (the bead or the capillary). give rise to the former spectrum, and is thus a nearly simultaneous Such a precise starting position should be rare, yet we measure of the background noise. This increase is due to stochastic observe this type of event frequently at low force. Further variations of the motion of the protein, and is proportional to the protein’s step size d. The line is a fit to the predicted increase evidence disfavoring multiple active motors is the appearance 2 2 (Charvin et al, 2002): y¼ d/vS/2p f þ b, where /vS is the mean of direction-reversal events, in which the velocities of descent velocity and b accounts for the (white) measurement noise. From and ascent are nearly equivalent (Figure 2A). This process is many such fits, we find that, if the FtsK enzymatic cycle has a 50C difficult to explain if two motors are acting, since both motors single rate-determining step, it advances 1272 bp per cycle. would have to switch directions simultaneously. For these reasons, we conclude that during each measured event, only one motor is translocating. 5 mM or forces between 5 and 19 pN. If we assume the active motor is a hexamer (Aussel et al, 2002), and that 100% of the Localizing the protein to a DNA extremity added protein in the bulk ATPase measurement were active, Although only one motor is active, loop formation requires we can use the maximum velocities of translocation the FtsK complex to contact the DNA in two locations. In 50C (B6.7 kbp/s) and ATP hydrolysis (B30 ATP/monomer/s) to what manner could the complex form a second, nontranslo- estimate that the FtsK motor moves B37 bp per hydro- cating contact? The protein could possibly have a second 50C lyzed ATP. Given the step size, this leads to the impossible (immobile) type of DNA-binding domain, but this is unlikely result that less than one ATP is hydrolyzed per enzymatic for an aforementioned reason: full-length, constant-velocity cycle. It is important to note that this does not invalidate our events require that the protein begins translocation at one of result for the lower bound of the step size: a much more likely the extremities of the DNA, and random DNA binding would explanation is that a significant fraction of the protein in the not efficiently localize the protein to an extremity. Instead, we ATPase assay was inactive; this would not affect the single- suggest that the second binding location is also a motor molecule measurement. bound to the DNA, but stalled at the bead or capillary surface, as diagrammed in Figure 6. In this scenario, a diffusing complex first binds to the DNA through one motor, which Discussion translocates (without loop formation or a change in bead FtsK forms loops of DNA height) and transports the complex to a surface, where the 50C We attribute the observed transient decreases in DNA exten- motor stalls but remains bound to the DNA. Other motors in sion (Figure 1B) to the extrusion of loops of DNA by the complex are then free to bind; translocation of these translocating protein complexes: a protein solely moving motors causes loop formation and reduces the bead height. along a DNA molecule will cause no change in extension in The complex is localized to the extremity of the DNA, as our assay, and since each molecule is nicked we rule out required to explain the measured full-length events. The rare shortening due to an accumulation of supercoils. Formation events with twice the normal velocity (see Supplementary of DNA loops has been postulated before to explain the data) occur when a second motor binds before the first has topological modification of DNA substrates by FtsK stalled, resulting in a change in bead height at twice the 50C complexes (Aussel et al, 2002; Ip et al, 2003). A recurrent single-motor rate. Finally, this model explains the multiple &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2435 | | Single-molecule studies of FtsK translocation OA Saleh et al independent biochemical steps (involving no motion along the DNA, but rather, for example, ATP binding or a change in the protein’s internal conformation). The mechanical step requires the protein to change the height of the bead by a distance d and perform a work against the force of Fd; thus, the kinetic barrier of that step will increase at high hindering forces, decreasing the rate. If, at zero force, the mechanical step is much faster than the biochemical step, the zero-force velocity v will depend only on the step size and the bio- chemical step rate. As the force increases, the mechanical step rate slows until it becomes comparable to the biochem- ical step rate. For small F, the biochemical step determines the rate (and the velocity equals v , independent of the force), while for large F the mechanical step determines the rate (and the velocity decreases with force). This description qualita- Figure 6 Diagram of proposed steps in loop formation by FtsK tively matches our data at 5 mM ATP. Furthermore, it is 50C complexes. (A) Free complexes, containing several identical (prob- consistent with the 1 mM ATP data, where no velocity ably hexameric) motors, diffuse into the vicinity of the DNA. (B) decrease is seen up to F¼ 29 pN. The decrease to 1 mM ATP The complexes bind the DNA through a single motor, which slows the ATP-binding process, thus slowing the rate of the translocates and carries the complex toward an extremity of the DNA without changing the bead height. (C) Upon reaching the biochemical steps. To make the mechanical step rate compar- surface, the motor stalls but does not unbind, allowing other motors able to this lowered biochemical rate (i.e., to see a velocity in the complex to bind and translocate. (D) Translocation of the decrease) will thus require a larger force than was needed at second motor extrudes a loop of DNA, and decreases the bead 5 mM ATP; we apparently do not reach this regime at the height at the single-motor velocity. The decrease can be halted by (E) unbinding of either motor, leading to a fast recovery of the bead maximum force of 29 pN utilized in the 1 mM data. height, or (F) reversal of the translocation direction, and a slow For assisting forces, our data notably deviate from the recovery of bead height. We believe that direction reversal proceeds above model and the measurements on RNA polymerase through unbinding of the translocating motor, followed by binding (Neuman et al, 2003); in both, there is a continuation of and translocation by an oppositely directed motor from the same complex (see Discussion). the constant-velocity plateau. Instead, we observe a clear increase in velocity for large assisting forces (Figure 3). The velocity increase is not likely due to a change in the enzy- timescales in the off-time distributions (see Supplementary matic turnover rate (since it is unlikely that assisting force data): short off-times occur when only one of the two motors would quicken the limiting biochemical processes), but unbinds, leaving the complex in close proximity to the DNA rather due to an increase in the mechanical step size. and thus enabling fast rebinding, while long off-times occur Supporting this, we note that the velocity is still dependent when both motors unbind, and rebinding is limited by free on ATP in this regime; thus, the biochemical processes are diffusion of the protein complexes. still rate-determining. However, since our data are sparse at large negative forces due to the rarity of direction reversals Brief motor unbinding accompanies direction reversal (inset, Figure 2A) and the decrease in event size (Figure 4A), As shown in the inset to Figure 2A, direction reversal is less we cannot rule out the existence of a completely separate likely at higher forces. This can be explained by assuming mechanism of forward motion. that direction reversal first involves unbinding of the translo- cating motor, followed immediately by binding of an oppo- Force dependence of binding statistics indicates sitely directed motor: the observed decrease in binding rate a distortion of the DNA with force (i.e., the increase in off-time, see Figure 4A) would The mean value of the on-time decreases exponentially with then explain the decrease in the probability of direction force (see Figure 4A) until, at high hindering force, protein reversal with force (inset, Figure 2A). The oppositely directed activity is limited by this parameter. The exponential depen- motor could, in principle, be an inverted configuration of the dence can be accounted for by assuming that the DNA length first motor, but we consider it more likely that it is a different when bound by FtsK is shorter than when free. We model 50C (but nearby) motor within the same complex. this effect as a two-state system (Evans and Ritchie, 1997; Rief et al, 1998), where the bound and unbound states are Considerations on the mechanochemistry of the FtsK separated by a transition state with a higher free energy. Any 50C motor DNA length change l between the bound and transition states The force–velocity relationships of FtsK (Figure 3) show a would require unbinding to perform a work Fl against the 50C clear plateau for small forces of either orientation, along with applied force. This would affect the mean on-time (the a decrease in velocity (at saturating 5 mM ATP) for large inverse of the mean unbinding rate) by a factor exp(Fl/ hindering forces. This behavior is qualitatively similar to both kT), consistent with our observations of an exponential theoretical predictions (Keller and Bustamante, 2000) and dependence on force. Based on the fitted exponential decay previous results obtained on RNA polymerase (Wang et al, constant of 11.370.9 pN, we estimate that unbinding of 1998; Neuman et al, 2003). The behavior is indicative of two FtsK causes the DNA length to increase by 50C types of sequential steps in the reaction that causes forward lX0.3670.03 nm (the estimate is a lower bound since the movement of the protein: a force-dependent mechanical step measurement is not sensitive to any further change in DNA (involving motion along the DNA), and one or more force- length between the transition and unbound state; see 2436 The EMBO Journal VOL 23 NO 12 2004 &2004 European Molecular Biology Organization | | Single-molecule studies of FtsK translocation OA Saleh et al Supplementary data). This length change is much smaller activities of FtsK and these other proteins will create a tension than the minimal 80 bp binding site required to stimulate in the DNA analogous to the force we apply to the bead in our FtsK ’s ATPase activity (Massey et al, 2004). Therefore, it is assay. Just as we observe (Figure 4B), this tension will 50C consistent only with a small bend in the DNA induced at the decrease the distance FtsK can travel before unbinding. protein’s binding site. FtsK motors that pump in the wrong direction will thus quickly fall off, allowing other, perhaps correctly oriented, Implications of direction reversal for models of FtsK motors to bind. Although the lack of structural data on FtsK polarity precludes affirmation, the formation of higher order com- It has been suggested that some chromosomal oligomeric plexes by FtsK and the resulting in vitro direction-reversal 50C sequences, with a high skew that inverts at dif, could direct activity might indicate that higher order complexes are also formed in vivo by FtsK, which would facilitate this process. the FtsK translocation process (Perals et al, 2000; Corre and Motors pumping in the correct direction will not create Louarn, 2002), just like Chi sequences alter the enzymatic properties of RecBCD complexes (Spies et al, 2003). In this tension in the DNA, and will translocate a correspondingly regard, direction reversal is surprising because it indicates greater distance. In this way, FtsK would clear any misplaced that FtsK can move in both directions on the same segment chromosomal loops that pass through the septum. 50C of DNA. Furthermore, we observe full-length direction-rever- Interestingly, this model could be applied to sporulation in B. subtilis, assuming that condensation of DNA in the pre- sal translocation events on a lambda DNA fragment of spore generates sufficient tension. approximately half the size of the phage genome; introduc- tion of a complete phage lambda genome has been shown to This force-rectified translocation model applies to segrega- perturb chromosome polarity in vivo (Corre et al, 2000). To tion of normal daughter chromosomes and to resolution of confirm this finding, we perform experiments on a fragment chromosome dimers. For normal chromosome segregation, of the E. coli genome that was shown to be polarized (Perals FtsK would simply accelerate the clearing process; both et al, 2000). Full-length translocation events after direction genetic and cellular biology data indicate that FtsK is not absolutely required for cohesion and mid-cell positioning of reversal are again observed (Figure 2B). We conclude that the oligomeric DNA motifs that polarize the E. coli and lambda the large terminus region of the E. coli chromosome (Capiaux genomes do not constitute absolute blocks to DNA transloca- et al, 2002; Corre and Louarn, 2002). In the presence of tion by FtsK . We cannot rule out the possibilities that their chromosome dimers, misplaced loops could still occur; FtsK 50C action is probabilistic, or that it requires the presence of the would clear these loops from the septum until there remained N-terminal or linker domains of FtsK. However, our data raise only the two chromosome-connecting strands that contain the dif sites required for XerC/D recombination. the possibility that DNA sequence information plays only an indirect role on polarity of translocation by FtsK. For exam- ple, an additional protein could bind to specific DNA se- FtsK ’s high velocity enables fast processing quences and block FtsK translocation, just like Tus binding to 50C of misaligned DNA Ter sites can stop replication forks (Kamada et al, 1996), or, FtsK ’s maximum velocity of B6.7 kbp/s makes it the rather than acting on FtsK, the DNA sequence could direct 50C fastest DNA-based motor protein yet measured. Speeds simi- progressive, oriented condensation of the nucleoids, thereby lar to our measurements have been estimated for the total imposing that the dif region is the last chromosomal section transfer rate of the B. subtilis genome into a nascent spore by to be moved by FtsK. SpoIIIE (Errington et al, 2001). As mentioned above, we do not expect FtsK to mobilize such a large fraction of the E. coli A force-rectified translocation model could impose genome, but rather a tangle of DNA loops. Unravelling such directionality a tangle could require loops to be successively processed Based on the observed sensitivity of the travelled distance to several times, as has been reported in vitro for replicative force (Figure 4B), we propose an alternate mechanism that catenanes (Ip et al, 2003); thus FtsK ’s high speed could be could account for in vivo FtsK directionality. The parameters 50C needed to complete multiple processing of misaligned loops of the exponential curve fit of mean on-time versus force quickly. (Figure 4A), along with the maximum speed, indicate that at zero force the FtsK complex will travel, on average, 50C B11 kbp per binding event. Since DNA translocation most likely involves the binding of two identical motor units Materials and methods (Figure 6), the zero-force mean distance for each motor is Protein, substrate DNA, and slide preparation then twice that of the complex, orB22 kbp. This is much less FtsK is purified as described in Aussel et al (2002). l DNA (bp 50C than the total length of the E. coli chromosome (4.5 Mbp). 25 882–45 679) is amplified by long-range PCR. E. coli chromosomal However, the chromosome is compacted in the cell and it is DNA (bp 8877–22 422 after the XerC-binding site of dif) is purified from plasmid pFC94 (Perals et al, 2000). DNA substrates are ligated highly probable that to clear the septum, FtsK works on DNA to biotin- and digoxigenin-modified DNA fragments, which are loops of sizes similar to the zero-force mean distance. prepared by PCR using cognate modified nucleotides. The resulting We propose that a septum-bound FtsK motor, upon bind- constructs are incubated with 4.5 mm diameter paramagnetic ing a chromosomal loop, can translocate in either direction. streptavidin beads and added to 1-mm square cross-section glass However, if pumping in the wrong direction, it will pull capillaries, which had been washed with 0.1 M NaOH, coated with SigmaCote, and sequentially incubated with solutions of anti- against the large fraction of condensed DNA and work against digoxigenin and bovine serum albumin (BSA). We insure that the other proteins in the nascent daughter cell that maintain the beads we use are bound to the capillary by a single, nicked DNA chromosome position, such as MukB and active next-genera- molecule, and not a single unnicked, molecule or multiple tion DNA replisomes (Sherratt, 2003). The counteracting molecules, as described in Strick et al (1996, 1998). &2004 European Molecular Biology Organization The EMBO Journal VOL 23 NO 12 2004 2437 | | Single-molecule studies of FtsK translocation OA Saleh et al Measurement of applied force and bead height slow recoveries (inset, Figure 2A), the number of slow recovery segments of a certain minimum size (typically 0.5 mm; less at higher The image of each bead is captured by a CCD camera at 60 Hz and processed, using custom-written software, to give the 3D bead forces) are counted, and divided by the total number of events of position. Before protein addition, we calibrate the apparatus for that size; the threshold is needed due to the difficulty in each bead by measuring the applied force as a function of magnet/ characterizing very short recoveries. For the fluctuation analysis, capillary separation distance. The applied force is measured by within a data set, only long (40.75 mm) events are analyzed. A monitoring the mean squared transverse fluctuations /dx S of the straight line is fit to, and subtracted from, each event to acquire the bead (see Figure 1A), and applying the equipartition theorem: difference at each time point. The mean variance from the fit line is 1 1 2 kT ¼ hdx iF=L, where L is the measured bead height (Strick et al, computed for each event; typically, the mean variance across all 2 2 1996). Microscope drift is removed by simultaneously tracking a events forms a compact distribution. Outliers from this distribution reference bead stuck to the capillary surface, and subtracting its (more than 2 standard deviations larger than the mean) sometimes height from that of the experimental bead. occur, and are considered to contain anomalous noise; thus, the corresponding events are discarded. Power spectra are then Addition of protein to the capillary calculated from the suitable events using standard algorithms. Experiments are performed in a buffer containing 10 mM MgCl , 10 mM Tris pH 7.9, 50 mM NaCl, 1 mM DTT, 0.01% Triton X-100, Bulk ATPase assay and ATP. In all, 5–10 ml of 200 nM FtsK monomers is added to 50C Reactions are performed with 100 nM of FtsK (monomer), 5 nM 50C 200 ml of buffer in the capillary, and gently mixed. of 3 kb supercoiled plasmid DNA, 1 nM [a P]ATP and cold ATP in 10 ml of 10 mM Tris–HCl, pH 7.9, 10 mM MgCl , 50 mM NaCl, and Long data acquisitions 1 mM DTT. Reactions are incubated for 3 min before being stopped Activity resulting from a single addition of protein can last for with EDTA and excess ATP. The ratio of ATP to ADP is analyzed by hours. To control for possible degradation of the protein or ATP in thin layer chromatography. We check that our measurements that time, we measure the activity at a reference force multiple correspond to initial rates and that DNA is in a 10-fold excess in times throughout the experiment. We never observe a decrease in the reaction (data not shown). velocity in those reference measurements. In some long measure- ments, we do observe an increase in the mean off-time; we attribute this to a decrease in the active protein concentration through Supplementary data degradation or nonspecific adsorption to the capillary. Data sets Supplementary data are available at The EMBO Journal Online. containing such a decrease are not used to construct the off-time plot in Figure 4A. Acknowledgements Data analysis Measured traces of bead height correspond directly to DNA We thank D Bensimon, V Croquette, J-M Louarn, and F Cornet for extension; no filtering has been applied to any of the plots shown. constant support and helpful discussions. We thank V Croquette for Translocation velocities, lengths, and on- and off-times are sharing analysis software, and J-Y Bouet, G Charvin, K Neuman, extracted by analyzing each data set with custom-written software and T Lionnet for critical reading of the manuscript. Research was that applies a filter (Chung and Kennedy, 1991) in order to identify funded by the CNRS and the Ecole Normale Supe´rieure. Research in events automatically, while still measuring each parameter from the Paris was supported by grants from the French Research Ministry unfiltered data. Data quoted in units of base pairs have been ACI Jeune Chercheur program and from the EU MolSwitch program. corrected for the difference between measured DNA extension and Research in Toulouse was supported by grants from the CNRS ATIP the true contour length by applying the worm-like chain model for program and from the French Research Ministry Fundamental DNA elasticity (Bouchiat et al, 1999). 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Journal

The EMBO Journal – Springer Journals

Published: Jun 16, 2004

Keywords: chromosome segregation; DNA translocation; FtsK; magnetic tweezers; molecular motor

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