RESUMEN
Nucleotide excision repair (NER) removes a wide variety of helix distorting DNA lesions. NER comprises two damage recognition sub-pathways: GG-NER operates genome wide, whereas TC-NER specifically removes transcription-blocking lesions from the transcribed strand of actively transcribed genes. NER is a multistep process, which requires the concerted action of 30 proteins that need to be tightly controlled at the right time and place for efficient repair. Post-translational protein modifications (PTMs) are common regulators of complex protein networks. Several NER factors were shown to be modified by ubiquitin, whereas others are actively involved in the ubiquitin-proteasome system itself. PTMs by ubiquitylation can be swiftly induced in a reversible manner and have the ability to regulate protein function, localization or stability. This makes the regulation by ubiquitin highly suitable for the coordination of the complex NER reaction. Accumulating evidence, including proteome wide quantitative proteomics approaches, showed that especially NER factors involved in the damage recognition are regulated by ubiquitin, emphasizing the high level of regulation during the initiation of the NER reaction. In this review we will therefore focus on the different functions of ubiquitylation during the DNA damage recognition steps of NER.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Ubiquitina/metabolismo , Animales , Humanos , Procesamiento Proteico-Postraduccional , Transcripción Genética , UbiquitinaciónRESUMEN
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.
Asunto(s)
Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular Tumoral , Daño del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Proteínas Nucleares/genética , ARN Interferente Pequeño , Proteína SUMO-1/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.
Asunto(s)
Reparación del ADN , Proteína de Replicación A/metabolismo , Ciclo Celular , Línea Celular/efectos de la radiación , Núcleo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN/efectos de la radiación , Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Cinética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Rayos UltravioletaRESUMEN
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.