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

Yoshitaka Kawasoe, Sakiko Shimokawa, Peter J. Gillespie, J. Julian Blow, Toshiki Tsurimoto, Tatsuro S. Takahashi

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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.
Original languageEnglish
Article number105588
JournalJournal of Biological Chemistry
Volume300
Issue number1
Early online date21 Dec 2023
DOIs
Publication statusPublished - Jan 2024

Keywords

  • DNA replication
  • Xenopus egg extract
  • clamp loader
  • mismatch repair
  • proliferating cell nuclear antigen
  • replication clamp

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