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

Yongqiang Lai,Wenwen Wu,Mingzhang Zhu,Tomohiko Ohta,Weixin Liang,Yukiko Togashi,Nana Rokutanda

doi:10.1038/s41598-019-50812-x

Yongqiang Lai, Wenwen Wu + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-50812-x

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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.

Highlights

One of the most noteworthy features of cancer cells is their genomic instability, which can largely be attributed to an aberrant response to replication stress; this is increased in cancer cells with activated oncogenes and accelerated cell growth
While RPA1 is a major subunit involved in DNA binding, both RPA2 and RPA3 are required for stability of the complex, and RPA2 is a major target for upstream kinases, including CDKs and the three phosphoinositide-3-kinase-related kinases (PIKKs) (ATR, ataxia–telangiectasia mutated [ATM], and DNA-PK)[6,7,8]
The results suggest that HERC2 interacts with Replication protein A (RPA) via its C-terminal HECT domain and that this interaction was enhanced by inhibition of proteasomes

Summary

Introduction

One of the most noteworthy features of cancer cells is their genomic instability, which can largely be attributed to an aberrant response to replication stress; this is increased in cancer cells with activated oncogenes and accelerated cell growth. ATR-mediated phosphorylation of RPA stimulates DNA synthesis and prevents ssDNA accumulation to alleviate low-level replication stress[15]. We show that the phosphorylation and protein stability of RPA2 are regulated by HERC2, a large HECT-type E3 ubiquitin ligase that plays critical roles in DNA replication and the damage response[16,17,18,19]. We build on those results by further defining the essential roles of HERC2 in the degradation and ATR-mediated Ser[33] phosphorylation of RPA2 in unstressed cells and cells under low-level replication stress. Our results show that HERC2 regulates 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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