Each of our cells is faced with as many as 10,000 DNA lesions per day. DNA repair mechanisms are crucial to remove lesions from our genomes in order to maintain genetic stability. The inability to remove lesions from the genome leads to mutations and unstable genomes, which can ultimately cause cancer and neurodegeneration.
The Nucleotide excision repair (NER) group was founded in 2015 to study this versatile DNA repair mechanism that removes a wide variety of structurally unrelated lesions from the human genome. Two mechanistically distinct NER pathways exist: a global genome repair pathway (GGR) that eliminates lesions throughout the genome and a transcription-coupled repair pathway (TCR) that removes lesions exclusively from actively transcribed DNA strands The clinical importance of this DNA repair mechanism is underscored by the variety of human disorders caused by inherited NER defects such as the cancer-prone Xeroderma pigmentosum (XP) disorder or Cockayne syndrome (CS), which causes severe neurodegeneration.
Model of NER complex assembly
Despite increasing insights, we still know very little about the regulation of this DNA repair mechanism. We employ a systematic cross-disciplinary approach to explore and identify regulatory mechanisms that control human nucleotide excision repair and its impact on human health.
Our research involves the following topics:
1. Proteomics approaches to identify new regulators of NER
To identify new regulators of NER, we systematically map the interactome of NER proteins involved in TCR and GGR by quantitative mass spectrometry in human cells.
2. Genetic screens to systemically map regulators of NER
To identify new genes involved in NER, we perform genetic siRNA-based screens. The aim is to systematically identify the genes that operate in NER.
3. Functional analysis of regulators in NER and human disease
The proteomics and genetics approaches have identified a number of new regulatory factors that play a role in NER. We will functionally dissect the role of these newly identified proteins by combining advanced microscopy techniques with innovative cell biological and biochemical approaches. To this end, we use patient-derived cell lines, as well as knock-down and knock-out cells generated using CRISPR-CAS9-based genome-editing techniques. Finally, iPS-based model systems are used to study mechanisms underlying neurodegeneration caused by NER defects. This will provide a framework to test drugs for their ability to mitigate neurological symptoms.
Below are some examples of the advanced microscopy approaches we employ to elucidate the mechanisms and regulation of NER.
Fluorescence loss in photobleaching (FLIP) to measure to dissociation-rate of YFP-tagged NER factor DDB2 (see Luijsterburg et al. J Cell Sci. 2007)
Chromatin unfolding by DDB2 visualized by tethering LacR-tagged DDB2 to a heterochromatic locus containing LacO (see Luijsterburg et al. J Cell Biol. 2012)
Forced chromatin tethering of DDB2 reveals an interaction with GFP-tagged DDB1 and CUL4A (see Luijsterburg et al. J Cell Biol. 2012)
Local UV-C irradiation triggers a local reduction in GFP-tagged histone H1 density at sites marked by DDB2-mCherry (see Luijsterburg et al. J Cell Biol. 2012)
Micro-irradiation of BrdU-sensitized cells with a pulsed UV-A laser triggers local recruitment of GFP-tagged XRCC1 and endogenous XRCC4 at sites marked by gH2AX (see Luijsterburg et al. Mol Cell. 2016)
Chromatin unfolding by RNF8 visualized by tethering LacR-tagged RNF8 to a heterochromatic locus containing LacO dependent on the chromatin remodeler CHD4 (see Luijsterburg et al. EMBO J. 2012)