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. 2010 Apr 21;29(8):1423-33.
doi: 10.1038/emboj.2010.29. Epub 2010 Apr 8.

Separating speed and ability to displace roadblocks during DNA translocation by FtsK

Estelle Crozat  1 , Adrien Meglio, Jean-François Allemand, Claire E Chivers, Mark Howarth, Catherine Vénien-Bryan, Ian Grainge, David J Sherratt
Affiliations

Separating speed and ability to displace roadblocks during DNA translocation by FtsK

Estelle Crozat et al. EMBO J. .

Abstract

FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism. Covalent trimers of wild-type subunits dimerized efficiently to form hexamers with high translocation activity and an ability to activate XerCD-dif chromosome unlinking. Covalent trimers with a catalytic mutation in the central subunit formed hexamers with two mutated subunits that had robust ATPase activity. They showed wild-type translocation velocity in single-molecule experiments, activated translocation-dependent chromosome unlinking, but had an impaired ability to displace either a triplex oligonucleotide, or streptavidin linked to biotin-DNA, during translocation along DNA. This separation of translocation velocity and ability to displace roadblocks is more consistent with a sequential escort mechanism than stochastic, hand-off, or concerted mechanisms.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
(A) Schematic of the FtsK proteins used. FtsK depicts the wild-type protein, with four transmembrane helices in the N-terminus region (dark green), a 639 amino-acid linker (blue line) and the C-terminus motor domain (light blue boxes) is drawn, containing the three subdomains α, β and γ. FtsK50C is the soluble E. coli FtsK derivative that has been used in previous in vitro studies. The pink box represents the 50 aa segment derived from the linker, which is directly linked to the C-terminal motor. The dark blue box is a segment absent in the FtsK50C structure and in the derivatives used here: monomer, covalent dimers and covalent trimers. Subunits in covalent multimer derivatives are connected by a 14 aa linker, joining directly the C-terminus of the γ-subdomain to the first amino acid of the motor, L840. (B) Different potential configurations for multimer formation. The 14 aa linker is sufficiently short that we expect subunit 1 (blue) to be always adjacent to 2 (yellow), and subunit 3 (green) adjacent to 2 in the trimers. Nevertheless, it is plausible that two types of trimers can form, with the subunits folding in either a clockwise, or anticlockwise sequence. These can then potentially form mixed hexamers (heterohexamers) or unmixed hexamers (homohexamers). Because of the uncertainty of the configuration in trimers, mutations were introduced into either subunit 2 (single mutants), or subunits 1 and 3 (double mutants). In parentheses, with a cross, is shown how three covalent trimers with a centrally placed mutated subunit could conceivably form a hexamer with wild-type subunits and three looped out mutated subunits. We have no evidence that this can form. (C) Mutant hexamers. This figure depicts the mutants used in this study. Pure hexamers are obtained with a wild-type, single-mutant trimer, double-mutant trimer or triple-mutant trimer (0, 2, 4 or 6). Mixes of trimers are required to form hexamers with 1, 3 or 5 mutant subunits (marked with an asterisk). Mutated subunits are yellow and wild-type subunits are blue.
Figure 2
Figure 2
In vitro activity of FtsK multimers. (A) ATP hydrolysis over time is shown for monomer, covalent dimer and trimer, at a concentration of 50 nM hexamer equivalent. The black arrow indicates the 1 min time point, which was used in later experiments (Figure 3). The mean data from three independent experiments, with standard deviations, are shown in panels (A–D). (B) FtsK-dependent XerCD-dif recombination. Recombination was followed over time on a dimeric plasmid containing two dif sites lacking consensus KOPS-loading sites. Proteins (50 nM hexamer equivalent) were incubated with ATP and pre-bound XerCD, and the 1 min time point data (arrow) were used to generate the data in Figure 3. (C) Triplex displacement as a function of time; protein concentration was 50 nM (hexamer equivalent); the dotted line shows FtsK50C activity as a comparison. (D) Triplex displacement as a function of protein concentration; 1 min reactions were used to compare translocation activity of monomer, dimers and trimers over a range of 0–350 nM (hexamer equivalent). The activity does not increase with higher concentrations of protein (data not shown). (E–G) Electron microscopy of covalent trimers of FtsK on DNA. (E) A 2.7 kb, linearized plasmid, was used to study DNA binding by the trimers. A representative field using wild-type trimer is shown, as well as an averaged image of the protein seen from the side (162 particles), showing that the particles have the overall size expected for a hexamer composed of two trimers. Similar results were obtained with double and triple WA mutant trimers (data not shown). The black bar represents 100 nm, and the white bar 13 nm. Arrows point at examples of particles and a schematic of the side-on view is drawn under the averaged reconstructed image (inset). (F) Wild-type trimers were incubated with a 44 bp DNA-containing KOPS, thereby leading to preferential top–bottom views, giving an averaged reconstruction of particles of ∼13 nm diameter (2600 particles selected) (inset). Similar results were obtained with double and triple WA mutant trimers (data not shown). Scale bars are the same as (E). The schematic indicates the orientation of the particle on the grid. (G) Examples of gold labelling. Two classes averaged from a 100 group classification are shown, with beads on opposite sides of the particle (left) or side-by-side (right).
Figure 3
Figure 3
In vitro activity of covalently linked mutated hexamers. (A) XerCD-dif recombination as a function of the number of mutated subunits. Recombination reactions were performed for 1 min on a dimeric plasmid containing two dif sites. In all, 50 nM hexamers were used on 27 nM 5.6 kb plasmid. % recombination is normalized to the activity of the wild-type (wt) hexamer, which gave ∼30% recombination in 1 min (Figure 2). The dotted line shows the levels if there were a linear activity decrease as a function of increasing number of mutated subunits (A–C). The panel below shows an example of a recombination gel (0.8% agarose, 1 × TAE) for hexamers containing 0–6 WB mutated subunits (S) denotes recombination substrate; (P) denotes product; (–) denotes no FtsK. Error bars indicate standard deviation in three independent experiments, and asterisks indicate the combinations made by mixing appropriate trimers as in Figure 1C (A–C). (B) ATPase activity measured on a fraction of the recombination reaction shown in (A). Activity is normalized to that of the wild-type hexamer. (C) Triplex displacement. Triplex-displacement assays were carried out as in Figure 2. After subtracting background, activity was normalized to that of the wild-type hexamer. The panel below shows an example of a triplex reaction gel (4% acrylamide, 1 × TAM) for hexamers containing 0–6 WB mutants.
Figure 4
Figure 4
Single-molecule analysis of covalent multimer translocation. Magnetic tweezers were used to study translocation of wild-type and double mutant hexamers, containing WA or WB in the central subunit of the trimers. Upper panels show examples of events obtained for each protein. There is no statistical significance to the relative lengths of the events shown. Light blue, data points; dark blue, fit to the data points. Lower panels show the distribution of average burst speed. The number of events, the average burst speed and its distribution are plotted under each panel. Green, data points, with s.d. (bars); red, Gaussian fits to the data. Note that the WA derivative shown here is biotin tagged and will, therefore, associate with the bead. A His-tagged derivative showed similar velocities. The data shown were obtained at a force of 5 pN and 2 mM ATP.
Figure 5
Figure 5
Displacement of the streptavidin ‘roadblock' during translocation. Streptavidin displacement activity was followed on a 597 bp biotinylated DNA. Excess streptavidin (wt, strong, or weak binding derivatives) was bound for 30 min and then excess free biotin was added. The indicated trimers (WA and WB contained mutations in the central subunit; 250 nM hexamer) were added, followed by ATP, and the reactions were stopped after 2 min. % displacement (determined by gel electrophoresis; 1.5% agarose/TBE; background subtracted) is indicated. Independent repetitions (⩾2) gave comparable results. (S) substrate; (P) product.
Figure 6
Figure 6
Translocation models. Scheme of FtsK translocation on DNA. The six subunits are drawn as spheres with a paddle representing the loop(s) in the α and/or β domains interacting with DNA nucleotides (black dots). One nucleotide is taken as a reference to show relative movement (yellow dot). Subunits inactivated by WA or WB catalytic mutations are indicated by a shorter paddle without the hook. Note that to simplify the drawing, subunits are represented linearly, but in reality would form a ring around DNA. (A) A sequential hand-off mechanism as applied to FtsK. In this model, all protein subunits change their local association with a given DNA segment, whether it be through specific contacts or not, during each chemical step (Massey et al, 2006; Enemark and Joshua-Tor, 2008). Each catalytic step leads to 2 bp dsDNA translocated. (B) Sequential escort model, as applied to FtsK. In this class of model, only one of the subunits changes its DNA contacts at each catalytic step and flexibility in the subunits and their loops allows movement of one subunit from the bottom to the top of the staircase during each catalytic step (Enemark and Joshua-Tor, 2008; Thomsen and Berger, 2009). In this panel, five of the six subunits are contacting every other nucleotide to avoid the substantial overwinding required to contact adjacent nucleotides. This model would require >30 Å movement of a subunit, as it moves from the bottom to the top of the staircase. The indicated nucleotide states of the subunits are; T, ATP bound; T*, ATP transition state; D, ADP bound; E, ‘exchange' (nucleotide free) (Thomsen and Berger, 2009). (C) Escort model with three subunit–nucleotide interactions, each occurring every other nucleotide. In this case, the movement of subunits and their loops during transition from the bottom to the top of the staircase would be similar to that for Rho/E1 (∼15 Å). (D) Escort model in a hexamer containing two mutated subunits. At least two protein-DNA contacts are still feasible in this situation, allowing velocity to remain the same, but reducing the work available to displace roadblocks.
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