Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Replication factor C (RFC) is crucial for DNA replication in eukaryotes and archaea.
• Archaeal RFC comprises small (RFCS) and large (RFCL) subunits homologous to eukaryotic counterparts.
• Understanding archaeal RFC structure provides insights into conserved replication mechanisms.

Purpose of the Study:

• To determine the oligomeric structure of the archaeal RFCS from Pyrococcus furiosus.
• To elucidate the three-dimensional (3D) structure of the RFCS hexamer.
• To investigate the functional role of RFCS in DNA synthesis.

Main Methods:

• Single-particle electron microscopy was employed to analyze RFCS oligomeric states.
• Random conical tilt reconstruction was used to obtain the 3D structure of the RFCS hexamer.
• In vitro assays measured the effect of RFCS and PCNA on DNA polymerase activity.

Main Results:

• RFCS predominantly forms hexameric rings at pH 9.0, and C-shaped tetramers/pentamers at pH 5.5.
• The 3D structure of the RFCS hexamer revealed a twisted ring with six subunits in a head-to-tail configuration.
• RFCS alone, with PCNA, significantly boosted DNA polymerase I activity, despite RFC typically requiring RFCL.

Conclusions:

• The determined 3D structure of RFCS provides a molecular basis for its organization and function.
• Archaeal RFCS can enhance DNA synthesis independently of RFCL, suggesting a flexible functional role.
• This study offers critical insights into the mechanism and functional states of RFC complexes in archaea.