Differential pathway control in nucleotide excision repair

Abstract

The stability and integrity of the genome is crucial for all cellular life on earth. This integrity is continuously challenged by internal and external genotoxic agents. These agents cause DNA damages which interfere with important cellular processes like replication of the genome and transcription of the genetic code. To protect the DNA against these agents, a complex network of dedicated DNA repair- and associated signalling pathways is in place. Collectively, these pathways are known as the DNA Damage Response (DDR).

NER is the main pathway for mammalian cells to remove UV-induced DNA lesions. The recognition of lesions in NER is either achieved by stalling of active RNA polymerase II during transcription or the stable binding of the protein XPC to the lesion. Although the majority of the factors involved in NER damage recognition have been identified, little is known about the molecular mechanisms and the regulation of this crucial step. The aim of the research described in this thesis focusses on the damage recognition, its regulation, and downstream effects in the NER pathway. Chapter I provides the needed background by introducing the current knowledge of the DDR pathways and their functioning. We discuss how proteins are modified and how this influences cellular signalling. Additionally we introduce different microscopy techniques and associated Monte Carlo modelling approaches used in this thesis. The Ubiquitin and SUMO protein modifications introduced in Chapter I, play key roles in cellular signalling pathways. In Chapter II we identify a new SUMO-targeted ubiquitin ligase (STUbL), RNF111/Arkadia. Utilizing its special SIM domains it specifically recognizes poly-SUMO chains on target proteins. Subsequently, it promotes non-proteolytic K63-linked ubiquitylation of the target protein using UBC13-MMS2 as the cognate E2 enzyme. RNF111 promotes ubiquitylation of the SUMOylated form of XPC, the damage sensor in NER. This ubiquitylation regulates the binding of XPC to the damaged DNA.

In Chapter III we go in further detail of what role the ubiquitylation of XPC by RNF111 plays in the NER pathway. We show that RNF111 is required for efficient repair of UV induced DNA lesions. Furthermore, the RNF111 mediated ubiquitylation promotes the release of XPC from the lesion after NER initiation. This release of XPC is needed to ensure stable incorporation of the NER endonucleases XPC and ERCC1/XPF. This sequential modification of XPC upon UV irradiation represents an extra layer of control of the NER reaction.

Under natural conditions, NER mostly operates at low damage levels, occasionally interrupted by higher levels, for instance by exposure to intense sunlight. Although highly relevant, knowledge on how cells respond to such fluctuations is sparse if not absent. In Chapter IV we show we show that cells switch between transcription-coupled repair (TCR) and global genome repair (GGR) dependent on damage load and therefore not only depend on the genomic location of the DNA lesion. At DNA damage concentrations below a threshold level, recruitment of the NER machinery by the global damage-sensor XPC is suppressed using regulatory ubiquitylation by E3 ligase Cul4a. Above threshold, damage-bound XPC switches to active recruitment of core NER factors. This bimodal switch allows cells under mild genotoxic stress to prioritize repair of the highly cytotoxic lesions that block transcription and are detected by RNA polymerase II in transcribed strands of active genes.

The bimodal switch described in chapter IV is very well suited to be described by Monte Carlo simulation. In Chapter V we use in silico experiments using in house modeling software to build on the experimental data. We hypothesized models with and without feedback loop systems governing this bimodality and see if damage marking can play a role. Furthermore we simulate what the effects are of competition for XPA RNA polymerase II (TCR) and XPC (GGR). This data combined is used to further elucidate the found regulatory mechanism and propose new in vivo experiments.