title: Distinct double- and single-stranded DNA binding of E. coli replicative DNA polymerase III alpha subunit.
abstract: The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.
authors.affiliation: Department of Physics, Northeastern University, Boston, Massachusetts, 02115, USA.
authors.forename: Micah J
authors.initials: MJ
authors.lastname: McCauley
authors.forename: Leila
authors.initials: L
authors.lastname: Shokri
authors.forename: Jana
authors.initials: J
authors.lastname: Sefcikova
authors.forename: Ceslovas
authors.initials: C
authors.lastname: Venclovas
authors.forename: Penny J
authors.initials: PJ
authors.lastname: Beuning
authors.forename: Mark C
authors.initials: MC
authors.lastname: Williams
mesh_terms: D004247:DNA / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding
chemical_list: D004277:DNA, Single-Stranded; D004247:DNA; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III