__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.
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