Fluorescence Measurements on the E. coli DNA Polymerase Clamp Loader: Implications for Conformational Changes during ATP and Clamp Binding

Eric R. Goedken, Marcia Levitus, Aaron Johnson, Carlos Bustamante, Mike O'Donnell, John Kuriyan

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Sliding clamps are ring-shaped proteins that tether DNA polymerases to their templates during processive DNA replication. The action of ATP-dependent clamp loader complexes is required to open the circular clamps and to load them onto DNA. The crystal structure of the pentameric clamp loader complex from Escherichia coli (the γ complex), determined in the absence of nucleotides, revealed a highly asymmetric and extended form of the clamp loader. Consideration of this structure suggested that a compact and more symmetrical inactive form may predominate in solution in the absence of crystal packing forces. This model has the N-terminal domains of the δ and δ′ subunits of the clamp loader close to each other in the inactive state, with the clamp loader opening in a crab-claw-like fashion upon ATP-binding. We have used fluorescence resonance energy transfer (FRET) to investigate the structural changes in the E. coli clamp loader complex that result from ATP-binding and interactions between the clamp loader and the β clamp. FRET measurements using fluorophores placed in the N-terminal domains of the δ and δ′ subunits indicate that the distances between these subunits in solution are consistent with the previously crystallized extended form of the clamp loader. Furthermore, the addition of nucleotide and clamp to the labeled clamp loader does not appreciably alter these FRET distances. Our results suggest that the changes that occur in the relative positioning of the δ and δ′ subunits when ATP binds to and activates the complex are subtle, and that crab-claw-like movements are not a significant component of the clamp loader mechanism.

Original languageEnglish (US)
Pages (from-to)1047-1059
Number of pages13
JournalJournal of molecular biology
Volume336
Issue number5
DOIs
StatePublished - Mar 5 2004

    Fingerprint

Keywords

  • ATPase
  • Clamp loader
  • DNA polymerase
  • DNA replication
  • Sliding clamp

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this