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 mismatch repair (MMR). Furthermore, Xenopus egg extracts have also been instrumental to dissect the DNA damage response, a complex signaling pathway including several protein kinases (e.g ATM, ATR, DNA PK, CHK1) and E3 ubiquitin ligases (e.g. FA core complex, BRCA1, RAD18, TRAIP and several Cullin E3 ligases).
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 by cutting-edge mass spectrometry.
DNA Repair in Xenopus Egg Extracts
Using a novel in vitro repair system, we showed that Xenopus egg extracts faithfully repair plasmid substrates containing a single DNA interstrand crosslink (ICL) Räschle et al. Cell 2009. Analysis of the DNA repair intermediates revealed the sequential steps of ICL repair. Convergence of two replication forks triggers dual incisions on either side of the crosslink in one of the parental strands Douwel et al. Mol. Cell 2014. The unhooked crosslink is further trimmed by nucleases and DNA synthesis resumes passed the lesion in a reaction catalyzed by specialized DNA repair polymerases. Finally the broken sister chromatide is repaired in a reaction involving homologous recombination Long et al. Science 2011. Consistent with this complex repair mechanism, bypass of DNA ICLs requires many factors, including the so-called Fanconi Anemia proteins Knipscheer et al. Science 2009. Given the crucial function of these repair factors in the maintenance genome stability, defects in many of their genes has been causally linked to monogenetic DNA repair disorders characterized by cancer predisposition and severe developmental disorders.
Towards a comprehensive DNA Repair Atlas
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.