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bioRxiv · 10.1101/2024.05.08.593111

Structure of the human CTF18 clamp loader bound to PCNA

Briola, G.·Tehseen, M.·Al-Amodi, A.·Nguyen, P. Q.·Savva, C. G.·Hamdan, S. M.·De Biasio, A.
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Abstract

Sliding clamps like PCNA are crucial processivity factors for replicative polymerases, requiring specific clamp loaders for loading onto DNA. The human alternative clamp loader CTF18-RFC interacts with the leading strand polymerase Pol {varepsilon} and loads PCNA onto primer/template DNA using its RFC pentameric module. Here, we provide a structural characterization of the human CTF18-RFC complex and its interaction with PCNA. Our cryo-EM data support that the Ctf8 and Dcc1 subunits of CTF18-RFC, which form the regulatory module interacting with Pol {varepsilon}, are flexibly tethered to the RFC module. A 2.9 [A] cryo-EM structure shows the RFC module bound to PCNA in an auto-inhibited conformation similar to the canonical RFC loader, marking the initial step of the clamp-loading reaction. The unique RFC1 (Ctf18) large subunit of CTF18-RFC, which based on the cryo-EM map shows high relative flexibility, is anchored to PCNA through an atypical low-affinity PIP box in the AAA+ domain and engages the RFC5 subunit using a novel {beta}-hairpin at the disordered N-terminus. We show that deletion of this {beta}-hairpin impairs the CTF18-RFC-PCNA complex stability, slows down clamp loading, and decreases the rate of primer synthesis by Pol {varepsilon}. Our research identifies distinctive structural characteristics of the human CTF18-RFC complex, providing insights into its role in PCNA loading and the stimulation of leading strand synthesis by Pol {varepsilon}.

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Briola, G., Tehseen, M., Al-Amodi, A., Nguyen, P. Q., Savva, C. G., Hamdan, S. M., De Biasio, A.. 2024-05-09. Structure of the human CTF18 clamp loader bound to PCNA. https://doi.org/10.1101/2024.05.08.593111

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