Research in our lab focuses on elucidating the mechanisms of Nucleotide Excision Repair, in relation to human health. 

Current projects in the lab include:

Novel regulators of the UV response (Hannes Lans, Ozge Aydin, Nils Wijgers)

Most essential proteins that make up the core machinery of NER have been identified. However, not much is known about the regulation and organization of the complete UV-induced DNA damage response in which NER plays a central role.  Using model systems such as C. elegans (see figure 1) and mammalian cell culture we try to unravel how NER efficiency is regulated and how it functions in developing organisms.

Using genetic screens in C. elegans, followed by functional analysis in mammalian cells, we aim at identifying and characterizing new proteins that function in the UV response. Furthermore, we use C. elegans to determine the role of different NER proteins and pathways to the development and DNA repair in different tissues within the animal. Figure 2 shows an example of the repair of UV-induced DNA lesions (CPDs, green) in germ cells (DAPI, blue).  This shows repair is impaired in C. elegans mutant germ cells which lack functional XPA-1 and RAD-23 proteins.

Regulation of NER using proteomics and live cell imaging (Jurgen Marteijn, (1) Petra Schwertman, (2)Loes van Cuijk and (3)Maria Tresini)

Despite detailed insight into the core NER process, little is known about its dynamic regulation and how chromatin remodeling and damage signaling is regulated. To investigate these regulatory pathways w e are currently employing quantitative proteomics and live cell imaging approaches to get a better understanding of these topics. (1) Recent reports indicate that post-translational protein modifications with ubiquitin play a key role in regulating the DNA damage response (DDR). Our main focus of this project is to unravel the role of the ubiquitin-proteasome-system during DNA-repair by studying the differences in ubiquitination after UV damage using quantitative proteomics. (2) Using live cell imaging in combination with our in house developed UV laser (266nm) we can study the in vivo dynamics of different GFP-tagged DDR proteins upon UV-C damage. (3) Combining the library of GFP-tagged NER proteins with quantitative interaction proteomics we will determine time-resolved changes in known and newly-discovered NER-regulators (NER-interactome). Thereby we will generate a better understanding of the regulation of NER and its connection with chromatin remodeling and damage signaling.

Analysis and diagnosis of patients with inherited NER defects (Koos Jaspers, Anja Raams)

The genetics of inherited NER deficiency are complex. Among affected patients, one of at least 12 different genes may be involved, and the type of mutation will determine the clinical outcome as either highly cancer-prone Xeroderma Pigmentosum or Cockayne syndrome and Trichothiodystrophy, without elevated cancer risk but features of accelerated ageing. The symptoms may vary from extremely mild with a life expectancy beyond 60 years to severe developmental failure and death within one year. In order to better understand the relation between DNA repair and human disease (cancer, ageing, neurodegeneration), but als o as a courtesy to affected families, our department offers worldwide specialist diagnostic service for patients suspected of a NER defect. Various aspects of NER activity are carefully characterised by state-of-the-art functional assays and the affected gene with its mutations is identified. This longstanding project has resulted in discovery of new repair disorders as well as genetic counselling and prenatal diagnostics. Figure 3 shows an example of a UDS complementation analysis experiment on Xeroderma Pigmentosum patient cell lines, which is used to determine defects in Nucleotide Excision Repair.

Herve Menoni

The molecular mechanism of NER on processing UV-lesions has been extensively studied, however the role of NER factors in oxidative DNA lesion removal is poorly understood. Our goal is to molecularly dissect the involvement of NER proteins, in the repair of oxidative DNA damages in vivo. Our first aim is to evaluate the recruitment of NER factors to oxidative DNA lesions generated in living cells by using confocal microscopy and a laser micro-irradiation generating specifically those lesions. How DNA repair of oxidative DNA lesions occurs in a chromatin context is also investigated.