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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: 9.81). 04/2012; 109(17):6531-6. DOI: 10.1073/pnas.1201613109
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ABSTRACT 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.
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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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