Summary
Endogenous and exogenous agents constantly damage DNA. Unrepaired lesions results in cell death and mutations, which may lead to accelerated aging or malignant transformation. A network of damage-response (DDR) mechanisms, notably DNA-repair, signalling, chromatin-remodelling, cell-cycle checkpoints and apoptosis collectively safeguard genomic integrity. Different DNA repair mechanisms constitute the first line of defence. The most versatile repair system is nucleotide excision repair (NER) that eliminates a wide spectrum of DNA injury, including UV-induced lesions. NER is intimately linked to transcription, replication and other repair processes. Despite detailed knowledge on each of these pathways little is known about the complex regulation between these processes. Recent reports indicate that post-translational protein modifications with ubiquitin play a key role in regulating the DNA damage response (DDR). To unravel the role of the ubiquitin-proteasome-system (UPS) in DDR we will investigate with complementary proteomic and genetic procedures:
A. Which proteins are ubiquitinated upon DNA-damage and what is the function of these modifications? Using an innovative proteomics approach, by combining dedicated cell-labelling procedures (SILAC) with ubiquitin purification techniques we aim to quantitatively identify novel and or differentially ubiquitinated proteins after UV-damage using tandem mass-spectrometry.
B. Which proteins regulate ubiquitination in DDR? To this aim we will perform a dedicated genetic screen testing all known E3 ubiquitin ligases and de-ubiquitinating enzymes for their role during DNA-repair using a novel high-throughput siRNA array.
The identified differentially ubiquitinated target proteins as well as UPS enzymes involved in DDR will provide novel insights into the molecular mechanism of age-related diseases or cancer and are attractive therapeutic targets.