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

Gabriella N. L. Chua; Emily C. Beckwitt; Victoria Miller-Browne; Olga Yurieva; Dan Zhang; Bryce J. Katch; Nina Y. Yao; John W. Watters; Kaitlin Abrantes; Ryogo Funabiki; Xiaolan Zhao; Mike E. O’Donnell; Shixin Liu

doi:10.1016/j.cell.2025.12.029

BiophysicsCellJan 28, 2026

Peer-reviewed journal article

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

Gabriella N. L. Chua iD, Emily C. Beckwitt iD, Victoria Miller-Browne iD, Olga Yurieva iD, Dan Zhang iD, Bryce J. Katch, Nina Y. Yao iD, John W. Watters iD, Kaitlin Abrantes, Ryogo Funabiki, Xiaolan Zhao iD, Mike E. O’Donnell iD, Shixin Liu iD

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Abstract

The ring-shaped sliding clamp 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 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 complex is required for processive DNA synthesis by polymerase {delta} (Pol{delta}), as the PCNA-Pol{delta} assembly is inherently unstable. This architectural role of RFC is dependent on the BRCT domain of Rfc1, and mutation of its DNA-binding residues causes sensitivity to DNA damage in vivo. We further showed the FEN1 flap endonuclease can also stabilize the PCNA-Pol{delta} interaction and mediate robust synthesis. Overall, our work revealed that, beyond their canonical enzymatic functions, PCNA-binding proteins harbor non-catalytic functions essential for DNA replication and genome maintenance.