Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2012; 109(17):6531-6. DOI: 10.1073/pnas.1201613109
Source: PubMed


FtsK is a hexameric DNA translocase that participates in the final stages of bacterial chromosome segregation. Here we investigate the DNA-binding and translocation activities of FtsK in real time by imaging fluorescently tagged proteins on nanofabricated curtains of DNA. We show that FtsK preferentially loads at 8-bp KOPS (FtsK Orienting Polar Sequences) sites and that loading is enhanced in the presence of ADP. We also demonstrate that FtsK locates KOPS through a mechanism that does not involve extensive 1D diffusion at the scale of our resolution. Upon addition of ATP, KOPS-bound FtsK translocates in the direction dictated by KOPS polarity, and once FtsK has begun translocating it does not rerecognize KOPS from either direction. However, FtsK can abruptly change directions while translocating along DNA independent of KOPS, suggesting that the ability to reorient on DNA does not arise from DNA sequence-specific effects. Taken together, our data support a model in which FtsK locates KOPS through random collisions, preferentially engages KOPS in the ADP-bound state, translocates in the direction dictated by the polar orientation of KOPS, and is incapable of recognizing KOPS while translocating along DNA.

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Available from: Estelle Crozat, Mar 10, 2014
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    • "Several studies have demonstrated that certain skewed sequences (i.e. sequences whose 385 presence is biased on the leading vs. the lagging strand) impart directionality to FtsK/SpoIIIE 386 translocation (Besprozvannaya et al., 2013; Cattoni et al., 2013; Lee et al., 2012; Levy et al., 387 2005; Löwe et al., 2008). It is tempting to imagine that the strand tracking mechanism presented 388 here could be used by SpoIIIE to read these skewed sequences (i.e. the SpoIIIE Recognition 389 Sequences, SRS). "
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    ABSTRACT: SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in B. subtilis. Here we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base-pair, we deduce that SpoIIIE's step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.
    eLife Sciences 10/2015; 4. DOI:10.7554/eLife.09224 · 9.32 Impact Factor
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    • "Clearly, FtsK-C can change direction while translocating DNA (see above and fig. 1 d). Locations of turn or pauses were precisely determined on a λ-DNA containing 3 KOPS [Lee et al., 2012]. This experiment showed that FtsK-C translocation reversal was independent of the presence of KOPS. "
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    ABSTRACT: A global view of bacterial chromosome choreography during the cell cycle is emerging, highlighting as a next challenge the description of the molecular mechanisms and factors involved. Here, we review one such factor, the FtsK family of DNA translocases. FtsK is a powerful and fast translocase that reads chromosome polarity. It couples segregation of the chromosome with cell division and controls the last steps of segregation in time and space. The second model protein of the family SpoIIIE acts in the transfer of the Bacillus subtilis chromosome during sporulation. This review focuses on the molecular mechanisms used by FtsK and SpoIIIE to segregate chromosomes with emphasis on the latest advances and open questions. © 2015 S. Karger AG, Basel.
    Journal of Molecular Microbiology and Biotechnology 04/2014; 24(5-6):396-408. DOI:10.1159/000369213 · 2.10 Impact Factor
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    • "Bacillus subtilis SpoIIIE and Escherichia coli FtsK are highly homologous motors responsible for the transfer of chromosomal DNA during cell division and sporulation. SpoIIIE and FtsK are able to translocate DNA rapidly both in vitro [4–7 kb/s at room temperature (1–3) and 17 kb/s at 37°C (4)] and in vivo [500 bp/s at 30°C (3)]. In sporulating B. subtilis, three steps are necessary for the newly replicated chromosome to be completely segregated into the forespore. "
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    ABSTRACT: SpoIIIE/FtsK are a family of ring-shaped, membrane-anchored, ATP-fuelled motors required to segregate DNA across bacterial membranes. This process is directional and requires that SpoIIIE/FtsK recognize highly skewed octameric sequences (SRS/KOPS for SpoIIIE/FtsK) distributed along the chromosome. Two models have been proposed to explain the mechanism by which SpoIIIE/FtsK interact with DNA. The loading model proposes that SpoIIIE/FtsK oligomerize exclusively on SpoIIIE recognition sequence/orienting polar sequences (SRS/KOPS) to accomplish directional DNA translocation, whereas the target search and activation mechanism proposes that pre-assembled SpoIIIE/FtsK hexamers bind to non-specific DNA, reach SRS/KOPS by diffusion/3d hopping and activate at SRS/KOPS. Here, we employ single-molecule total internal reflection imaging, atomic force and electron microscopies and ensemble biochemical methods to test these predictions and obtain further insight into the SpoIIIE-DNA mechanism of interaction. First, we find that SpoIIIE binds DNA as a homo-hexamer with neither ATP binding nor hydrolysis affecting the binding mechanism or affinity. Second, we show that hexameric SpoIIIE directly binds to double-stranded DNA without requiring the presence of SRS or free DNA ends. Finally, we find that SpoIIIE hexamers can show open and closed conformations in solution, with open-ring conformations most likely resembling a state poised to load to non-specific, double-stranded DNA. These results suggest how SpoIIIE and related ring-shaped motors may be split open to bind topologically closed DNA.
    Nucleic Acids Research 12/2013; 42(4). DOI:10.1093/nar/gkt1231 · 9.11 Impact Factor
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