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bioRxiv · 10.1101/2023.08.28.555229

The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts

Kawasoe, Y.·Shimokawa, S.·Gillespie, P. J.·Blow, J. J.·Tsurimoto, T.·Takahashi, T. S.
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Abstract

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.

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Kawasoe, Y., Shimokawa, S., Gillespie, P. J., Blow, J. J., Tsurimoto, T., Takahashi, T. S.. 2023-08-30. The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts. https://doi.org/10.1101/2023.08.28.555229

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