Structure of the human CTF18−RFC clamp loader bound to PCNA

Overview

This study presents a structural characterization of the human CTF18–RFC clamp loader complex and its interaction with PCNA, the sliding clamp processivity factor essential for replicative DNA polymerases. CTF18–RFC represents an alternative clamp loader to the canonical RFC complex, specialized for loading PCNA on the leading strand during DNA replication. The complex comprises two functional modules: a catalytic RFC module responsible for PCNA loading, containing the Ctf18 large subunit and four smaller RFC subunits (RFC2-5), and a regulatory module formed by Ctf8 and Dcc1 subunits that interacts with the leading strand polymerase Pol ε. While CTF18–RFC is not essential for bulk DNA replication in yeast, it plays critical roles in sister chromatid cohesion and replication checkpoint activation. Previous structural work has characterized the regulatory module's interaction with the catalytic domain of Pol ε, but the architecture of the RFC module and its engagement with PCNA remained incompletely understood. This research addresses that gap through cryo-electron microscopy analysis of the human CTF18–RFC complex bound to PCNA, revealing distinctive structural features that distinguish this alternative loader from canonical RFC.

Methods and approach

The study employed cryo-electron microscopy to determine the structure of the human CTF18–RFC complex in association with PCNA. A high-resolution 2.9 Å cryo-EM structure was obtained showing the RFC module bound to PCNA. The cryo-EM data were analyzed to assess the conformational states and flexibility of different subunits and modules within the complex. To probe the functional significance of structural features, deletion mutagenesis was performed targeting a novel β-hairpin element identified at the N-terminus of the Ctf18 subunit. Biochemical assays were conducted to evaluate the effects of this deletion on CTF18–RFC−PCNA complex stability and clamp-loading kinetics. Additional functional assays measured the impact on primer synthesis rates by Pol ε to assess how structural alterations in CTF18–RFC affect its stimulation of leading strand synthesis.

Results

The cryo-EM analysis revealed that the Ctf8 and Dcc1 subunits of the regulatory module are flexibly tethered to the RFC catalytic module, consistent with predictions of a flexible linker connecting these functional segments. The 2.9 Å structure showed the RFC module bound to PCNA in an autoinhibited conformation resembling that of the canonical RFC loader, representing the initial step of the clamp-loading reaction pathway. The unique Ctf18 large subunit exhibited high relative flexibility in the cryo-EM map and was found to anchor to PCNA through an atypical low-affinity PIP box motif located in the AAA+ domain. A novel β-hairpin structure was identified at the disordered N-terminus of Ctf18 that mediates engagement with the RFC5 subunit. Deletion of this β-hairpin impaired the stability of the CTF18–RFC−PCNA complex, reduced the rate of clamp loading, and decreased the rate of primer synthesis by Pol ε, demonstrating its functional importance for both structural integrity and catalytic efficiency of the complex.

Implications

The structural characterization identifies distinctive features of the human CTF18–RFC complex that differentiate it from the canonical RFC loader while revealing conserved aspects of the clamp-loading mechanism. The autoinhibited conformation of the RFC module bound to PCNA suggests that CTF18–RFC follows the established three-step clamp-loading pathway, but employs specialized structural elements for its specific functions in leading strand replication. The atypical low-affinity PIP box and the novel β-hairpin element represent unique adaptations of the Ctf18 subunit that may contribute to the functional specialization of this alternative loader. The flexible tethering of the regulatory module supports a model in which the Ctf18-1-8 segment can independently engage Pol ε while the RFC module performs PCNA loading, enabling coordination between polymerase recruitment and clamp loading at the leading strand. The demonstration that deletion of the β-hairpin affects both complex stability and Pol ε activity indicates that structural features of CTF18–RFC directly influence its stimulation of leading strand synthesis. These findings advance understanding of how alternative clamp loaders achieve functional specialization through structural variations on a conserved catalytic framework, with implications for models of leading strand replication dynamics and the coordination of PCNA loading with polymerase processivity.