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

High fidelity and efficiency are critical for chromosomal DNA duplication in eukaryotes. This precision is achieved through the coordinated action of replicative DNA polymerases, Pol δ and Pol ε, anchored to the sliding clamp, proliferating cell nuclear antigen (PCNA) (Hubscher et al., 2002). The ring-shaped PCNA homotrimer encircles DNA, enhancing the processivity of these polymerases in DNA replication and repair processes, including excision repair and homologous recombination (Choe and Moldovan, 2017), and plays an important role in sister chromatid cohesion establishment (Moldovan et al., 2006). The closed-ring topology of PCNA necessitates the transient opening of one interface to facilitate the incorporation and trapping of primer–template DNA. This critical function is executed by pentameric AAA+ (ATPases Associated with diverse cellular Activities) ATPases, collectively known as clamp loaders (Indiani and O’Donnell, 2006; Kelch et al., 2012; Kang et al., 2019). Extensive structural and biochemical work in different organisms (Jeruzalmi et al., 2001; Bowman et al., 2004; Kazmirski et al., 2004; Gaubitz et al., 2020; Gaubitz et al., 2022; Schrecker et al., 2022) established a common mechanism for the clamp-loading reaction, which proceeds through three main steps (Turner et al., 1999; Gomes et al., 2001; Chen et al., 2009; Simonetta et al., 2009; Kelch et al., 2011; Kelch, 2016). Initially, the clamp loader engages with the intact clamp ring, adopting an autoinhibited configuration that precludes DNA binding (Gaubitz et al., 2020). This stage sets the groundwork for the subsequent transition, where the clamp loader alters its conformation to an activated state. This change facilitates the opening of the clamp ring, creating a gap sufficiently large to permit the passage of duplex DNA and aligning the clamp loader for primer–template DNA interaction. The final phase of this process is initiated by the binding of DNA to the clamp loader, catalysing ATP hydrolysis. DNA binding prompts the closure of the clamp and hydrolysis of ATP induces the concurrent disassembly of the closed clamp loader from the sliding clamp–DNA complex, completing the cycle necessary for the engagement of the replicative polymerases to start DNA synthesis.

The canonical replication factor C (RFC), consisting of a large RFC1 subunit and four subunits (RFC2, RFC3, RFC4, and RFC5), acts as the primary clamp loader for PCNA (Indiani and O’Donnell, 2006; Jeruzalmi et al., 2002). However, eukaryotes also employ alternative loaders (Lee and Park, 2020), including CTF18–RFC (Kang et al., 2019; Mayer et al., 2001; Naiki et al., 2001; Bylund and Burgers, 2005; Crabbé et al., 2010), which likely use a conserved loading mechanism but are functionally specialized through specific protein interactions and context-dependent roles in DNA replication.

Genome-wide analyses revealed that CTF18–RFC predominantly facilitates PCNA loading on the leading strand, in contrast to the canonical RFC complex, which mainly loads PCNA on the lagging strand (Liu et al., 2020). Although CTF18–RFC is not essential for bulk DNA replication in yeast (al-Khodairy and Carr, 1992; Kim et al., 2005), it plays a vital role in ensuring sister chromatid cohesion (Mayer et al., 2001; Bermudez et al., 2003a; Terret et al., 2009). This may be due to its selective loading of PCNA linked to EcoI acetyltransferase (Moldovan et al., 2006), which acetylates Smc3, preventing cohesin destabilization by WapI (Zhang et al., 2008; Rolef Ben-Shahar et al., 2008; Unal et al., 2008). Recruitment of CTF18–RFC to the leading strand is also important for activation of the replication checkpoint (Naiki et al., 2001; Crabbé et al., 2010; Kubota et al., 2013; Stokes et al., 2020).

The CTF18–RFC complex features two distinct modules with separate functions (Bylund and Burgers, 2005; Bermudez et al., 2003a; Bermudez et al., 2003b; Fujisawa et al., 2017): a catalytic RFC module for PCNA loading, consisting of a large RFC1-like subunit (hereby referred to as Ctf18), alongside the four RFC2, RFC3, RFC4, and RFC5 subunits utilized by the canonical RFC complex, and a regulatory module, made up of the Ctf8 and Dcc1 subunits. These latter two subunits attach to the Ctf18 C-terminus, forming the Ctf18-1-8 module. A long linker predicted to be flexible connects these two segments (Stokes et al., 2020; Grabarczyk et al., 2018). Structural and functional studies have demonstrated that the interaction between the Ctf18-1-8 module and the catalytic domain of Pol2 (Pol2CAT), the principal subunit of Pol ε, directs CTF18–RFC to replication forks and boosts its clamp-loading efficiency (Stokes et al., 2020; Grabarczyk et al., 2018). This interaction situates the clamp loader close to the primer/template junction, facilitating PCNA loading (Stokes et al., 2020). Pol2CAT is tethered to the remainder of Pol ε via an unstructured linker, making it an integral component of the CMGE (Cdc45-Mcm-GINS-Pol ε) core replisome complex (Jones et al., 2021). It has been proposed that Pol ε utilizes both the CMG helicase and PCNA as processivity factors to facilitate normal replication rates (Langston et al., 2014; Yeeles et al., 2017). Tethering by the CMG complex might allow Pol ε to dissociate from the 3′-end of the leading strand but stay at the replication fork until leading strand synthesis restarts. Given the relatively weak interaction of Pol ε with PCNA (Chilkova et al., 2007), it is possible that multiple PCNA loading events on the leading strand via CTF18–RFC are necessary to achieve full Pol ε processivity.

Although the structure of the Ctf18-1-8 module in association with Pol2CAT has been elucidated (Stokes et al., 2020; Grabarczyk et al., 2018), the architecture of the CTF18–RFC module and its interaction with PCNA remain uncharacterized. To investigate the clamp-loading mechanism employed by CTF18–RFC, we reconstituted the full human CTF18–RFC complex with PCNA and determined its structure using cryo-electron microscopy (cryo-EM). Our cryo-EM data supports the prediction that the Ctf18-1-8 and RFC modules are flexibly tethered. The analysis yielded high-resolution reconstructions of the entire CTF18–RFC module bound to a closed PCNA ring. Although the overall architecture mirrors the autoinhibited state observed in the canonical RFC loader in a complex with PCNA (Gaubitz et al., 2020), distinctive features within CTF18–RFC were identified. These distinctions may offer insights into the specialized functional roles CTF18–RFC plays in DNA replication.