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

• Published in: Scientific reports Vol. 9; no. 1; pp. 14257 - 12
• Main Authors: Lai, Yongqiang, Zhu, Mingzhang, Wu, Wenwen, Rokutanda, Nana, Togashi, Yukiko, Liang, Weixin, Ohta, Tomohiko
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 03.10.2019; Nature Publishing Group UK
• Subjects: Cell proliferation; Deoxyribonucleic acid; DNA; DNA helicase; Epistasis; Genomes; Guanine Nucleotide Exchange Factors - metabolism; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; Kinases; Phosphorylation; Protein Interaction Maps; Protein Subunits - metabolism; Proteolysis; Replication protein A; Replication Protein A - metabolism; Single-stranded DNA; siRNA; Ubiquitin; Ubiquitin - metabolism; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-50812-x

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.