Multistep loading of a DNA sliding clamp onto DNA by replication factor C

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Proliferating cell nuclear antigen (PCNA) is a crucial sliding clamp for eukaryotic DNA replication and repair.
• Replication factor C (RFC) is an ATP-dependent clamp loader responsible for loading PCNA onto DNA at specific sites like nicks and 3' single-stranded/double-stranded DNA junctions.

Purpose of the Study:

• To elucidate the mechanism of PCNA loading by RFC.
• To investigate the structural dynamics of RFC:PCNA complexes bound to DNA substrates.

Main Methods:

• Cryogenic electron microscopy (cryo-EM) was used to determine the structures of RFC:PCNA complexes.
• Structures were resolved for complexes bound to ATPγS and DNA substrates, including 3' single-stranded/double-stranded DNA junctions and nicked DNA.

Main Results:

• Observed unexpected open and closed conformations of PCNA within the RFC:PCNA:DNA complex.
• Demonstrated that PCNA can adopt an open, planar state facilitating direct double-stranded DNA insertion.
• Identified a role for the Rfc1 N-terminal BRCT domain in coordinating DNA prior to insertion into the RFC central channel.

Conclusions:

• PCNA ring closure may not be directly coupled to ATP hydrolysis.
• Partial DNA melting facilitates PCNA's lateral insertion into the RFC:PCNA complex.
• A revised model for RFC-catalyzed PCNA loading is proposed, highlighting the importance of DNA substrate conformation and the Rfc1 domain.