Chua Gabriella N L, Beckwitt Emily C, Miller-Browne Victoria, Yurieva Olga, Zhang Dan, Katch Bryce J, Watters John W, Abrantes Kaitlin, Funabiki Ryogo, Zhao Xiaolan, O'Donnell Michael E, Liu Shixin
Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, New York, NY, USA.
Laboratory of DNA Replication, The Rockefeller University, New York, NY, USA.
bioRxiv. 2025 Aug 12:2025.08.08.669392. doi: 10.1101/2025.08.08.669392.
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 δ (Polδ), as the PCNA-Polδ 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-Polδ 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.
环形滑动夹增殖细胞核抗原（PCNA）能使DNA聚合酶在复制和修复过程中进行持续性DNA合成。PCNA加载到DNA上是由ATP酶夹加载器RFC催化的。利用单分子平台来可视化PCNA与RFC在DNA上的动态相互作用，我们意外地发现，与传统观点相反，加载后RFC仍继续与PCNA结合。在功能上，这种夹加载器/夹复合物是聚合酶δ（Polδ）进行持续性DNA合成所必需的，因为PCNA-Polδ组装本质上是不稳定的。RFC的这种结构作用依赖于Rfc1的BRCT结构域，其DNA结合残基的突变会导致体内对DNA损伤敏感。我们进一步表明，FEN1瓣内切核酸酶也能稳定PCNA-Polδ相互作用并介导稳健的合成。总体而言，我们的工作表明，除了其典型的酶功能外，PCNA结合蛋白还具有对DNA复制和基因组维持至关重要的非催化功能。
