title: Single-molecule atomic force spectroscopy reveals that DnaD forms scaffolds and enhances duplex melting.
abstract: The Bacillus subtilis DnaD is an essential DNA-binding protein implicated in replication and DNA remodeling. Using single-molecule atomic force spectroscopy, we have studied the interaction of DnaD and its domains with DNA. Our data reveal that binding of DnaD to immobilized single molecules of duplex DNA causes a marked reduction in the 'end-to-end' distance of the DNA in a concentration-dependent manner, consistent with previously reported DnaD-induced looping by scaffold formation. Native DnaD enhances partial melting of the DNA strands. The C-terminal domain (Cd) of DnaD binds to DNA and enhances partial duplex melting but does not cause DNA looping. The Cd-mediated melting is not as efficient as that caused by native DnaD. The N-terminal domain (Nd) does not affect significantly the DNA. A mixture of Nd and Cd fails to recreate the DNA looping effect of native DnaD but produces exactly the same effects as Cd on its own, consistent with the previously reported failure of the separated domains to form DNA-interacting scaffolds.
authors.affiliation: Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
authors.forename: Wenke
authors.initials: W
authors.lastname: Zhang
authors.forename: Cristina
authors.initials: C
authors.lastname: Machón
authors.forename: Alberto
authors.initials: A
authors.lastname: Orta
authors.forename: Nicola
authors.initials: N
authors.lastname: Phillips
authors.forename: Clive J
authors.initials: CJ
authors.lastname: Roberts
authors.forename: Stephanie
authors.initials: S
authors.lastname: Allen
authors.forename: Panos
authors.initials: P
authors.lastname: Soultanas
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020029:Base Pairing; D004269:DNA, Bacterial / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C092895:DnaD protein, Bacillus subtilis