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.