Differences between bacteria and eukaryotes in clamp loader mechanism, a conserved process underlying DNA replication

J Biol Chem. 2024 Apr;300(4):107166. doi: 10.1016/j.jbc.2024.107166. Epub 2024 Mar 14.


Clamp loaders are pentameric ATPases that place circular sliding clamps onto DNA, where they function in DNA replication and genome integrity. The central activity of a clamp loader is the opening of the ring-shaped sliding clamp and the subsequent binding to primer-template (p/t)-junctions. The general architecture of clamp loaders is conserved across all life, suggesting that their mechanism is retained. Recent structural studies of the eukaryotic clamp loader replication factor C (RFC) revealed that it functions using a crab-claw mechanism, where clamp opening is coupled to a massive conformational change in the loader. Here we investigate the clamp loading mechanism of the Escherichia coli clamp loader at high resolution using cryo-electron microscopy. We find that the E. coli clamp loader opens the clamp using a crab-claw motion at a single pivot point, whereas the eukaryotic RFC loader uses motions distributed across the complex. Furthermore, we find clamp opening occurs in multiple steps, starting with a partly open state with a spiral conformation, and proceeding to a wide open clamp in a surprising planar geometry. Finally, our structures in the presence of p/t-junctions illustrate how the clamp closes around p/t-junctions and how the clamp loader initiates release from the loaded clamp. Our results reveal mechanistic distinctions in a macromolecular machine that is conserved across all domains of life.

Keywords: AAA + ATPase; DNA replication; bacteria; clamp loader; cryo-EM.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Cryoelectron Microscopy
  • DNA Replication*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Models, Molecular
  • Protein Conformation
  • Protein Structure, Quaternary
  • Replication Protein C / chemistry
  • Replication Protein C / genetics
  • Replication Protein C / metabolism