Mechanism of an ATP-fueled molecular motor that transports DNA across membranes

How is DNA transported across membranes? We investigated the mechanism of DNA segregation by a bacterial DNA motor that uses the energy of ATP hydrolysis to power the movement of chromosomes across membranes during bacterial cell division.

HFSP Career Development Award holder Marcelo Nollmann and colleagues
authored on Mon, 15 July 2013

Our model system is the process of DNA transport across the membrane of sporulating Bacillus subtilis by the SpoIIIE motor. SpoIIIE assembles into an hexameric ring and pumps DNA by following hundreds of specific DNA sequences (called SRS) that are scattered across the chromosome and serve as ‘sign posts’ for SpoIIIE to know in which direction it should move. The size of the SpoIIIE hexameric motor is such that it allows only the transport of a single DNA molecule across the division membrane, thus wire-stripping all DNA-bound factors (including RNA polymerase!) and resetting the transcriptional program between the two cellular compartments. In two studies, we investigated the architecture of the DNA translocation complex in live cells and the mechanism by which SpoIIIE interacts with SRS.

In our first study, we investigated the architecture of the SpoIIIE motor by super-resolution microscopy in live cells. Recently, microscopy and cryo-EM experiments suggested that SpoIIIE forms a channel through which DNA is transported. We tested the predictions of this model by photo-activated localization microscopy, structured illumination microscopy and fluorescence fluctuation microscopy. These methods allowed us to obtain unprecedented detail of the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We found that SpoIIIE assembles into ~45 nm complexes containing 47 ± 20 SpoIIIE molecules. These complexes are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. Finally, we showed that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with a previous model proposing that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, where SpoIIIE coordinates completion of chromosome segregation with membrane fusion.

Figure 1: Three dimensional super-resolution image illustrating the compartment-specific localization of the SpoIIIE complex (green) while translocating the chromosome across the sporulation septum (red) in a live B. subtilis cell.

Previous models have suggested that SpoIIIE load on SRS sequences to bias the direction of movement of the motor (loading model). We used a novel combination of ensemble and single-molecule methods to dissect the series of steps required for SRS localization and motor activation by SpoIIIE. We showed that SpoIIIE/DNA association kinetics are sequence-independent with binding specificity being uniquely determined by dissociation. Next, we employed single-molecule and computational methods to demonstrate that hexameric SpoIIIE binds DNA non-specifically and finds SRS by an ATP-independent target search mechanism with interactions between SpoIIIE and SRS triggering allosteric, orientation-specific activation of the motor. These results allowed us to refute the SRS loading model and to propose a conceptually novel mechanism that involves three sequential steps (see figure): (i) binding of pre-formed hexamers to non-specific DNA, (ii) specific localization of SpoIIIE to SRS by target search exploration involving either or both sliding and/or hopping, and (iii) sequence-specific activation of the motor mediated by binding and oligomerization of SpoIIIE on SRS.

  

Figure 2: a) Atomic force micrograph of SpoIIIE-DNA complexes. The orange arrow indicates SpoIIIE bound to non-specific DNA sequences and green arrow SpoIIIE complexes that reached SRS sequences. b) Schematic representation of the target search and sequence-specific activation model. SpoIIIE pre-formed hexamers bind to non-specific DNA and by target search exploration reach SRS sequences, that will, in turn, stimulate the motor activity.

References

1) Recruitment, Assembly, and Molecular Architecture of the SpoIIIE DNA Pump Revealed by Superresolution Microscopy. Fiche JB, Cattoni DI, Diekmann N, Langerak J, Clerte C, Royer C, Margeat E, Doan T, Nöllmann* M.  PLoS Biol. 2013 May;11(5):e1001557.

2) SpoIIIE mechanism of directional translocation involves target search coupled to sequence-dependent motor stimulation. Cattoni D.I., Chara O., Godefroy C., Margeat E., Trigueros S., Milhiet P.E., Nöllmann* M. EMBO Rep, 2013 May 2;14(5):473-9. doi:10.1038/embor.2013.39.

Pubmed link (Ref 1)

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