title: The Escherichia coli clamp loader can actively pry open the β-sliding clamp.
abstract: Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245.
authors.forename: Christopher O
authors.initials: CO
authors.lastname: Paschall
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Thompson
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245.
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Marzahn
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245.
authors.forename: Ankita
authors.initials: A
authors.lastname: Chiraniya
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245.
authors.forename: Jaclyn N
authors.initials: JN
authors.lastname: Hayner
authors.affiliation: Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021.
authors.forename: Mike
authors.initials: M
authors.lastname: O'Donnell
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245.
authors.forename: Arthur H
authors.initials: AH
authors.lastname: Robbins
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245.
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245. Electronic address: lbloom@ufl.edu.
authors.forename: Linda B
authors.initials: LB
authors.lastname: Bloom
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000379:methods; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D015233:Models, Statistical; D008968:Molecular Conformation; D009154:Mutation; D011487:Protein Conformation
chemical_list: D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D001120:Arginine; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases