ABSTRACT
XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4(DDB2) ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial for efficient progression of the NER reaction, thereby providing an extra layer of quality control of NER.
Subject(s)
DNA Repair/physiology , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Nuclear Proteins/metabolism , SUMO-1 Protein/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line, Tumor , DNA Damage , DNA-Binding Proteins/genetics , Endonucleases/genetics , Endonucleases/metabolism , Humans , Nuclear Proteins/genetics , RNA, Small Interfering , SUMO-1 Protein/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13-Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response.
Subject(s)
DNA Repair/physiology , Nuclear Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation/physiology , Ubiquitin-Protein Ligases/metabolism , Ubiquitins/metabolism , Cell Line, Transformed , DNA Damage/physiology , HeLa Cells , Humans , Ligases/genetics , Ligases/metabolism , Nuclear Proteins/genetics , Plasmids/genetics , RNA, Small Interfering/genetics , Signal Transduction/physiology , Signal Transduction/radiation effects , Small Ubiquitin-Related Modifier Proteins/genetics , Sumoylation/radiation effects , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination/physiology , Ubiquitination/radiation effects , Ubiquitins/genetics , Ultraviolet Rays/adverse effectsABSTRACT
Protein ubiquitylation has emerged as a key regulatory mechanism in DNA-damage signalling and repair pathways. We report a proteome-wide, site-specific survey of ubiquitylation changes after ultraviolet irradiation, identifying numerous upregulated and downregulated ubiquitylation sites on known components of DNA-damage signalling, as well as on proteins not previously implicated in this process. Our results uncover a critical role for PCNA-associated factor PAF15 (p15(PAF)/KIAA0101) ubiquitylation during DNA replication. During unperturbed S phase, chromatin-associated PAF15 is modified by double mono-ubiquitylation of Lys 15 and 24 templated through PCNA binding. Replication blocks trigger rapid, proteasome-dependent removal of Lys 15/24-ubiquitylated PAF15 from PCNA, facilitating bypass of replication-fork-blocking lesions by allowing recruitment of translesion DNA synthesis polymerase polη to mono-ubiquitylated PCNA at stalled replisomes. Our findings demonstrate widespread involvement of ubiquitin signalling in genotoxic-stress responses and identify a critical function for dynamic PAF15 ubiquitylation in safeguarding genome integrity when DNA replication is challenged.
