title: SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination.
abstract: Translesion DNA synthesis (TLS) is a universal DNA damage tolerance mechanism conserved from yeast to mammals. A key event in the regulation of TLS is the monoubiquitination of proliferating cell nuclear antigen (PCNA). Extensive evidence indicates that the RAD6-RAD18 ubiquitin-conjugating/ligase complex specifically monoubiquitinates PCNA and regulates TLS repair. However, the mechanism by which the RAD6-RAD18 complex is targeted to PCNA has remained elusive. In this study, we used an affinity purification approach to isolate the PCNA-containing complex and have identified SIVA1 as a critical regulator of PCNA monoubiquitination. We show that SIVA1 constitutively interacts with PCNA via a highly conserved PCNA-interacting peptide motif. Knockdown of SIVA1 compromised RAD18-dependent PCNA monoubiquitination and Polη focus formation, leading to elevated ultraviolet sensitivity and mutation. Furthermore, we demonstrate that SIVA1 interacts with RAD18 and serves as a molecular bridge between RAD18 and PCNA, thus targeting the E3 ligase activity of RAD18 onto PCNA. Collectively, our results provide evidence that the RAD18 E3 ligase requires an accessory protein for binding to its substrate PCNA.
authors: Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors: Jinhua
authors: J
authors: Han
authors: Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors: Ting
authors: T
authors: Liu
authors: Department of Anatomy and Center for Cancer Research, The University of Hong Kong, Hong Kong, China Department of Anatomy and Center for Cancer Research, The University of Hong Kong, Hong Kong, China.
authors: Michael S Y
authors: MS
authors: Huen
authors: Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors: Lin
authors: L
authors: Hu
authors: Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors: Zhiqiu
authors: Z
authors: Chen
authors: Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China jhuang@zju.edu.cn.
authors: Jun
authors: J
authors: Huang
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D001665:Binding Sites; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D025941:Protein Interaction Mapping; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination; D014466:Ultraviolet Rays
chemical_list: D051017:Apoptosis Regulatory Proteins; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C485954:RAD18 protein, human; C106549:SIVA1 protein, human; D044767:Ubiquitin-Protein Ligases