1. Academic Validation
  2. HERC2 regulates RPA2 by mediating ATR-induced Ser33 phosphorylation and ubiquitin-dependent degradation

HERC2 regulates RPA2 by mediating ATR-induced Ser33 phosphorylation and ubiquitin-dependent degradation

  • Sci Rep. 2019 Oct 3;9(1):14257. doi: 10.1038/s41598-019-50812-x.
Yongqiang Lai 1 2 Mingzhang Zhu 1 2 Wenwen Wu 1 Nana Rokutanda 1 3 Yukiko Togashi 1 Weixin Liang 2 Tomohiko Ohta 4
Affiliations

Affiliations

  • 1 Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.
  • 2 Department of General Surgery, The People's Hospital of Gaoming District of Foshan City, Foshan city, Guangdong province, China.
  • 3 Oncology TA Division/Research & Development, AstraZeneca Japan, Osaka, Japan.
  • 4 Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan. to@marianna-u.ac.jp.
Abstract

Replication protein A (RPA) binds to and stabilizes single-stranded DNA and is essential for the genome stability. We reported that an E3 ubiquitin Ligase, HERC2, suppresses G-quadruplex (G4) DNA by regulating RPA-helicase complexes. However, the precise mechanism of HERC2 on RPA is as yet largely unknown. Here, we show essential roles for HERC2 on RPA2 status: induction of phosphorylation and degradation of the modified form. HERC2 interacted with RPA through the C-terminal HECT domain. Ubiquitination of RPA2 was inhibited by HERC2 depletion and rescued by reintroduction of the C-terminal fragment of HERC2. ATR-mediated phosphorylation of RPA2 at Ser33 induced by low-level replication stress was inhibited by depletion of HERC2. Contrary, cells lacking HERC2 catalytic residues constitutively expressed an increased level of Ser33-phosphorylated RPA2. HERC2-mediated ubiquitination of RPA2 was abolished by an ATR Inhibitor, supporting a hypothesis that the ubiquitinated RPA2 is a phosphorylated subset. Functionally, HERC2 E3 activity has an epistatic relationship with RPA in the suppression of G4 when judged with siRNA knockdown experiments. Together, these results suggest that HERC2 fine-tunes ATR-phosphorylated RPA2 levels through induction and degradation, a mechanism that could be critical for the suppression of secondary DNA structures during cell proliferation.

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