A non-catalytic role for RFC in PCNA-mediated processive DNA synthesis
Gabriella N L Chua1,2,3, Emily C Beckwitt2,3, Victoria Miller-Browne4,5
1Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, New York, NY, USA.
Biorxiv : the Preprint Server for Biology
|August 20, 2025
Summary
The clamp loader RFC stays with PCNA after loading, which is crucial for stable DNA synthesis by Polymerase Delta. This unexpected role of RFC is vital for genome maintenance.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Proliferating Cell Nuclear Antigen (PCNA) is essential for DNA replication and repair, enabling processive DNA synthesis.
- Replication Factor C (RFC) is the ATPase clamp loader responsible for loading PCNA onto DNA.
Purpose of the Study:
- To investigate the dynamic interaction between RFC and PCNA on DNA using single-molecule visualization.
- To elucidate the functional significance of the RFC-PCNA complex in DNA synthesis.
Main Methods:
- Single-molecule platform for dynamic visualization of protein-DNA interactions.
- Site-directed mutagenesis to study the role of specific RFC domains (BRCT domain).
- In vivo assays to assess DNA damage sensitivity.
Main Results:
- RFC remains associated with PCNA after loading, challenging previous models.
- The RFC-PCNA complex is essential for the processive DNA synthesis by Polymerase Delta (Polδ) due to inherent instability of the PCNA-Polδ assembly.
- The BRCT domain of Rfc1 mediates this architectural role, and its DNA-binding residues are critical.
- Flap endonuclease 1 (FEN1) also stabilizes the PCNA-Polδ interaction.
Conclusions:
- RFC and other PCNA-binding proteins possess non-catalytic functions critical for DNA replication and genome stability.
- The continued association of RFC with PCNA is a key architectural feature supporting processive DNA synthesis.
- Understanding these non-canonical roles is essential for comprehending DNA replication and repair mechanisms.
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