Ubiquitin-Mediated Regulation of Damage Recognition in Nucleotide Excision Repair

__Abstract__ __Scope of the thesis:__ The integrity of our genetic information is continuously threatened by endogenous metabolites and environmental agents that can generate a variety of DNA lesions. Accumulation of DNA damage can induce genetic changes or cell death, which may result in the onset of cancer or premature ageing. To deal with these adverse effects a network of DNA repair mechanisms and damage signaling pathways, known as the DNA Damage Response (DDR), has evolved. Nucleotide Excision Repair (NER) is responsible for the repair of a wide variety of helix distorting lesions, including those induced by UV-light. NER is a multistep process, which requires the action of more than 30 proteins that need to be tightly controlled to function at the right time and place to warrant efficient repair. Complex cellular processes are commonly regulated by various post translational modifications. Most notably, protein ubiquitylation has emerged as a key regulator of NER. The aim of the work presented in this thesis is to understand the regulation and dynamic properties of NER factors and the UV-DDR in general by ubiquitylation. Chapter 1 provides the necessary background and the current knowledge on ubiquitinmediated regulation of the DNA damage recognition steps of NER. Mass spectrometry (MS) can be used to study the ubiquitylation status of proteins on a proteome wide scale. Since ubiquitylation is transient and solely occurs on a small fraction of proteins, methods to enrich for these proteins are required to study them with MS. In chapter 2, a quantitative proteomics approach was combined with the isolation of ubiquitylated peptides to identify UV-regulated ubiquitylation sites on proteins. In addition to the well-known ubiquitylated NER factors, XPC and DDB, we identified UV-responsive ubiquitylated proteins that are active in different biological pathways including, DNA repair, chromatin remodeling, transcription, mRNA splicing, translation and the ubiquitin proteasome system. The most UVinduced peptides were identified for Histone H1. UV-induced H1 ubiquitylation was validated by biochemical experiments. Chapter 3 describes the identification and characterization of a new ubiquitin E3 ligase - RNF111 - required for efficient NER. RNF111 belongs to the class of SUMO-targeted ubiquitin ligases (STUbLs), which provide direct crosstalk between SUMOylation and ubiquitylation. RNF111 specifically recognizes proteins modified with poly-SUMO2/3 chains, and promotes UBC13-dependent K63-linked ubiquitylation. We demonstrate that RNF111 targets SUMOylated XPC, a DNA damage recognition factor in NER. In chapter 4 we have studied the function of the RNF111 mediated XPC ubiquitylation in vivo. Using a combination of DNA repair assays, immunofluorescence and live cell imaging experiments, we show that RNF111-mediated ubiquitylation stimulates the release of XPC from DNA lesions. This step is required for the stable incorporation of the NER endonucleases XPG and ERCC1/XPF to efficiently complete the NER reaction. In chapter 5 we further focus on XPC dynamics. In contrast to other NER factors, XPC shows a non-linear immobilization in response to increasing UV-doses. XPC binding is inhibited at low UV-C doses (0-4 J/m2), which is dependent on Cul4a and XPC ubiquitylation. NER comprises two damage recognition sub-pathways: global genome NER (GG-NER), involving XPC, and transcription coupled NER (TC-NER). We propose a model in which cells switch between suppression of stable binding of XPC to DNA lesions at low UV-doses and release of this inhibition at higher doses. This bimodal switch allows cells to prioritize repair of transcription blocking DNA lesions under mild genotoxic stress (low UV-dose, ≤ 4 J/m2). Chapter 6 describes the dynamic behavior of RPA in replication and NER. In contrast to other replication factors, RPA does not cluster in replication foci due to a very short residence time at single stranded DNA. During NER, RPA is involved in both the pre- and post-incision steps of NER. RPA binding to the pre-incision complex could only be visualized in the absence of incision without a substantial increase in residence time. Our data show that RPA is an intrinsically highly dynamic protein. In chapter 7 the main findings of this thesis are wrapped up and the perspectives derived from these data to obtain a more in depth view on the regulation of NER are being discussed

DNA damage-induced histone H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway

The DNA damage response (DDR), comprising distinct repair and signalling pathways, safeguards genomic integrity. Protein ubiquitylation is an important regulatory mechanism of the DDR. To study its role in the UV-induced DDR, we characterized changes in protein ubiquitylation following DNA damage using quantitative di-Gly proteomics. Interestingly, we identified multiple sites of histone H1 that are ubiquitylated upon UV-damage. We show that UV-dependent histone H1 ubiquitylation at multiple lysines is mediated by the E3-ligase HUWE1. Recently, it was shown that poly-ubiquitylated histone H1 is an important signalling intermediate in the double strand break response. This poly-ubiquitylation is dependent on RNF8 and Ubc13 which extend pre-existi

Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair

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RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response

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

RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response

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