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TitleDifferences between bacteria and eukaryotes in clamp loader mechanism, a conserved process underlying DNA replication.
Journal, issue, pagesJ Biol Chem, Vol. 300, Issue 4, Page 107166, Year 2024
Publish dateMar 13, 2024
AuthorsJacob T Landeck / Joshua Pajak / Emily K Norman / Emma L Sedivy / Brian A Kelch /
PubMed AbstractClamp 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 ...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.
External linksJ Biol Chem / PubMed:38490435
MethodsEM (single particle)
Resolution3.0 - 7.7 Å
Structure data

EMDB-43094, PDB-8val:
Structure of the E. coli clamp loader bound to the beta clamp in a Open-DNAp/t conformation
Method: EM (single particle) / Resolution: 3.7 Å

EMDB-43095, PDB-8vam:
Structure of the E. coli clamp loader bound to the beta clamp in a Semi-Open conformation
Method: EM (single particle) / Resolution: 3.9 Å

EMDB-43096, PDB-8van:
Structure of the E. coli clamp loader bound to the beta clamp in an Initial-Binding conformation
Method: EM (single particle) / Resolution: 7.7 Å

EMDB-43098, PDB-8vap:
Structure of the E. coli clamp loader bound to the beta clamp in a Fully-Open conformation
Method: EM (single particle) / Resolution: 3.0 Å

EMDB-43099, PDB-8vaq:
Structure of the E. coli clamp loader bound to the beta clamp in a Closed-DNA1 conformation
Method: EM (single particle) / Resolution: 3.8 Å

EMDB-43100, PDB-8var:
Structure of the E. coli clamp loader bound to the beta clamp in a Closed-DNA2 conformation
Method: EM (single particle) / Resolution: 3.9 Å

EMDB-43101, PDB-8vas:
Structure of the E. coli clamp loader bound to the beta clamp in an Altered-Collar conformation
Method: EM (single particle) / Resolution: 3.8 Å

EMDB-43102, PDB-8vat:
Structure of the E. coli clamp loader bound to the beta clamp in a Open-RNAp/t conformation
Method: EM (single particle) / Resolution: 3.2 Å

Chemicals

ChemComp-ZN:
Unknown entry

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

ChemComp-MG:
Unknown entry

Source
  • escherichia coli (E. coli)
  • synthetic construct (others)
KeywordsREPLICATION / TRANSFERASE/DNA / Bacterial Clamp Loader Complex / TRANSFERASE-DNA complex / TRANSFERASE / REPLICATION/DNA / REPLICATION-DNA complex

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