Mechanism of Sliding Clamp Loading During DNA Replication and Repair in Atomic Detail

Abstract

DNA replication is a fundamental process that is essential for all forms of life, and it is made efficient by ring-shaped sliding clamp proteins. One such protein is the eukaryotic sliding clamp, Proliferating Cellular Nuclear Antigen (PCNA), which not only facilitates replication but also coordinates multiple cellular pathways, such as DNA repair, cell cycle regulation, and apoptosis. The proper function of PCNA is critical for maintaining genome stability, making it a crucial factor in human health. The clamp loader complex is the primary regulator of sliding clamp activity. Replication Factor C (RFC), the eukaryotic clamp loader, is responsible for opening the closed PCNA and loading it onto DNA. However, the mechanism by which RFC accomplishes this task has been elusive for years. Our research has contributed to this field by revealing multiple cryo-electron microscopy (cryo-EM) structures of the RFC:PCNA complex that describe the steps involved in the clamp loading reaction. Specifically, we found that RFC opens PCNA with a 'crab-claw' motion, allowing it to preferentially bind to PCNA before DNA. Additionally, during replication, primer-template DNA, which is RFC's primary DNA substrate, directly binds to the central chamber of the complex. Our study also sheds light on the mechanism by which RFC performs its role in loading PCNA during DNA repair. When RFC binds to gapped or nicked DNA during DNA repair, it uses an additional DNA binding site, and both sites work together to melt the DNA strands with their 'separation pins.' This discovery provides the first structural insight into how RFC accomplishes its crucial functions in DNA replication and repair. Overall, our findings provide detailed atomic-level insights into how RFC efficiently loads PCNA onto different DNA substrates, advancing our understanding of this essential biological process.