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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    • "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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    ABSTRACT: Interest for proteins of the FtsK family initially arose from their implication in many primordial processes in which DNA needs to be transported from one cell compartment to another in eubacteria. In the first section of this chapter, we address a list of the cellular functions of the different members of the FtsK family that have been so far studied. Soon after their discovery, interest for the FstK proteins spread because of their unique biochemical properties: most DNA transport systems rely on the assembly of complex multicomponent machines. In contrast, six FtsK proteins are sufficient to assemble into a fast and powerful DNA pump; the pump transports closed circular double stranded DNA molecules without any covalent-bond breakage nor topological alteration; transport is oriented despite the intrinsic symmetrical nature of the double stranded DNA helix and can occur across cell membranes. The different activities required for the oriented transport of DNA across cell compartments are achieved by three separate modules within the FtsK proteins: a DNA translocation module, an orientation module and an anchoring module. In the second part of this chapter, we review the structural and biochemical properties of these different modules.
    Advances in Experimental Medicine and Biology 01/2013; 767:245-62. DOI:10.1007/978-1-4614-5037-5_12 · 1.96 Impact Factor
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