A non-catalytic role for RFC in PCNA-mediated processive DNA synthesis

The ring-shaped sliding clamp proliferating cell nuclear antigen (PCNA) enables DNA polymerases to perform processive DNA synthesis during replication and repair. The loading of PCNA onto DNA is catalyzed by the ATPase clamp-loader replication factor C (RFC). Using a single-molecule platform to visualize the dynamic interplay between PCNA and RFC on DNA, we unexpectedly discovered that RFC continues to associate with PCNA after loading, contrary to the conventional view. Functionally, this clamp-loader/clamp (CLC) complex is required for processive DNA synthesis by polymerase ẟ (Polẟ), as the PCNA-Polẟ assembly is inherently unstable. This architectural role of RFC is dependent on the BRCA1 C-terminal homology (BRCT) domain of Rfc1, and mutation of its DNA-binding residues causes sensitivity to genotoxic stress in vivo. We further showed that flap endonuclease I (FEN1) can also stabilize the PCNA-Polẟ interaction and mediate robust synthesis. Overall, our work revealed that, beyond their canonical enzymatic functions, PCNA-binding proteins harbor non-catalytic functions important for DNA replication and genome maintenance.