Proteomics of DNA repair
DNA repair processes are indispensable for maintaining genome stability. During S-phase, DNA repair processes enable the replication machinery to bypass DNA lesions. Inactivation of these processes leads to developmental abnormalities and cancer (e.g. Fanconi Anemia). Combining mass spectrometry based proteomics with powerful in vitro DNA repair systems we identify repair factors across DNA repair pathways and characterize their function at the molecular level. We would like to understand, how the repair processes are coupled to DNA replication and how the activity of the repair enzymes is tightly controlled and guided towards the DNA lesions.
Our Model System
To study DNA repair we use cell-free extracts derived from eggs of the African clawed frog Xenopus laevis. These extracts support efficient replication, condensation and segregation of chromosomal DNA, making them an ideal system to study chromatin-associated processes. They faithfully recapitulate several replication associated DNA repair pathways including the repair of DNA interstrand crosslinks (ICLs), DNA protein crosslinks (DPCs) or DNA mismatches (MMR). In this fully soluble system, damaged DNA is efficiently replicated in a highly synchronized manner. Quick isolation procedures allow to extract the replication and repair intermediates together with the associated proteins. While biochemical assays can be used to monitor progression of repair at the DNA level, the corresponding repair factors can be identified comprehensively by cutting-edge mass spectrometry.
Using comprehensive proteomic profiling we have gained unprecedented insight into the temporal recruitment of known and novel DNA repair factors involved in bypassing psoralen DNA crosslinks. While some of these factors contribute to accurate repair of the lesions others play important roles in DNA damage signaling. Using established DNA repair assays we will pinpoint the exact role of these factors. Through expanding the palette of DNA lesions, our long-term goal is to gain a global view of DNA repair. Using additional proteomic techniques (mapping of phospho- or ubiquitylation sites) we aim to uncover the underlying regulatory mechanism of various repair pathways.