The Escherichia coli Clamp Loader Can Actively Pry Open the β-Sliding Clamp

• Published in: The Journal of biological chemistry 2011-12, Vol.286 (49), p.42704-42714
• Main Authors: Paschall, Christopher O., Thompson, Jennifer A., Marzahn, Melissa R., Chiraniya, Ankita, Hayner, Jaclyn N., O'Donnell, Mike, Robbins, Arthur H., McKenna, Robert, Bloom, Linda B.
• Format: Article
• Language: English
• Subjects: AAA+ ATPase; Adenosine Triphosphatases - chemistry; Adenosine Triphosphate - chemistry; Arginine - chemistry; ATPases; Clamp Loader; Crystallography, X-Ray - methods; DNA and Chromosomes; DNA Polymerase; DNA Replication; DNA-Directed DNA Polymerase - chemistry; Dose-Response Relationship, Drug; Enzyme Kinetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Fluorescence; Kinetics; Microscopy, Fluorescence - methods; Models, Molecular; Models, Statistical; Molecular Conformation; Mutation; Protein Conformation; Sliding Clamp
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.268169

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.