title: Structure and DNA-binding properties of the Bacillus subtilis SpoIIIE DNA translocase revealed by single-molecule and electron microscopies.
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.
authors.affiliation: Centre de Biochimie Structurale, Department of Single-Molecule Biophysics CNRS UMR5048, INSERM U554, Université de Montpellier I & II, 29 rue de Navacelles, 34090 Montpellier, France.
authors.forename: Diego I
authors.initials: DI
authors.lastname: Cattoni
authors.forename: Shreyasi
authors.initials: S
authors.lastname: Thakur
authors.forename: Cedric
authors.initials: C
authors.lastname: Godefroy
authors.forename: Antoine
authors.initials: A
authors.lastname: Le Gall
authors.forename: Josephine
authors.initials: J
authors.lastname: Lai-Kee-Him
authors.forename: Pierre-Emmanuel
authors.initials: PE
authors.lastname: Milhiet
authors.forename: Patrick
authors.initials: P
authors.lastname: Bron
authors.forename: Marcelo
authors.initials: M
authors.lastname: Nöllmann
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C030069:spore-specific proteins, Bacillus; D004247:DNA