Cellular responses to DNA damage and replication stress
1 PhD project proposal in the IPP summer call 2019
To maintain genome stability, human cells rely on accurate DNA replication in S-phase and chromosome segregation in mitosis. Dynamic assembly and disassembly of protein-protein interactions and their regulation by posttranslational modifications is central for these processes. The genome is exposed to exogenous and endogenous insults that can induce a variety of lesions in different cell cycle stages. DNA lesions present in S-phase of the cell cycle can block the progression of replication forks and thus lead to replication stress. Replication stress refers to slow down or stalling of replication forks that can occur as consequence of exogenous DNA damage such as chemotherapeutic drugs as well as in response to endogenous sources: For instance, the progression of replication forks is problematic at chromosomal fragile sites, telomeres, repetitive sequences and DNA–RNA hybrids (e.g. R loops). Prolonged replication stress can also lead to the formation of highly cytotoxic DNA double strand breaks. Therefore, the mechanisms that protect stalled replication forks and repair replication stress-associated double strand breaks are essential for maintaining genome stability and preventing the development of diseases such as cancer.
PhD project: Analyses of the DNA replication stress response using mass spectrometry-based proteomics
The recruitment of DNA repair factors to sites of DNA lesions and replication stress is a dynamic process that is tightly regulated by posttranslational modifications of proteins, in particular phosphorylation and ubiquitylation. The full scope of proteins operating at different types of DNA lesions, in particular sites of replication stress, and the mechanisms that lead to the regulated recruitment of factors to these lesions are not understood. The PhD candidate will employ recently developed proximity tagging approaches based on engineered ascorbate peroxidase (APEX) or biotin ligase (BioID) combined with SILAC-based quantitative mass spectrometry to investigate the recruitment of proteins and the formation of protein complexes at sites of replication stress and replication stress-associated double strand DNA breaks. These studies will be performed after replication stress-induced by endogenous sources such as R-loops and exogenous sources such as chemotherapeutic drug hydroxyurea. Furthermore, wild type cells and cells lacking relevant protein kinases and ubiquitin ligases will be used to examine the impact of phosphorylation and ubiquitylation on protein dynamics on damaged chromatin. Proteomics experiments will be complemented by cell biological methods such as high throughput microscopy, flow cytometry, functional DNA repair assays and next generation sequencing. In addition, the PhD candidate will have the opportunity to collaborate with leading researchers in the field having complementary expertise in order to execute parts of the project. This projects aims to obtain a first systematic view of protein components and mechanisms that operate in the replication stress response and thus confer essential roles in genome stability maintenance.
Publications relevant to the project
Borisova ME, Voigt A, Tollenaere MAX, Sahu SK, Juretschke T, Kreim N, Mailand N, Choudhary C, Bekker-Jensen S, Akutsu M, Wagner SA, Beli P (2018). p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage. Nat Commun, 9:1017.
Wagner SA, Oehler H, Voigt A, Dalic D, Freiwald A, Serve H, Beli P (2016). ATR inhibition rewires cellular signaling networks induced by replication stress. Proteomics, 16, 402-16.
Yang J, Wagner SA, Beli P (2015). Illuminating Spatial and Temporal Organization of Protein Interaction Networks by Mass Spectrometry-Based Proteomics. Front Genet, 6, 344.
Schmidt CK, Galanty Y, Sczaniecka-Clift M, Coates J, Jhujh S, Demir M, Cornwell M, Beli P and Jackson SP (2015). Systematic E2 screening reveals a UBE2D-RNF138-CtIP axis promoting DNA repair. Nat Cell Biol, 17, 1458-70