Functional analysis of polyubiquitin chain linkage in genome maintenance

Abstract

Ubiquitin is a small post-translational modifier involved in almost all cellular processes in eukaryotes. The versatility of ubiquitin as a signaling molecule derives mainly from its ability to form ubiquitin chains of varying topologies. This work was set up to deepen our knowledge of the so far poorly understood function-topology relationship of different ubiquitin linkages. Thereby, we focused on the two most studied linkages, K48 and K63, and their biological involvement. The first project of this thesis describes the characterization of a tool to study these specific ubiquitin chains, while the second project focuses on deciphering the role of a specific K63-linked ubiquitin chain in DNA damage bypass by investigating a potential reader of this specific ubiquitin code. The first project "Specific binders for recognition, inhibition, and tracking of K48- and K63-polyubiquitin chain signaling" describes the design and characterization of specific affinity probes on the basis of designed ankyrin repeat proteins (DARPins) for the recognition, inhibition, or tracking of linkage-specific polyubiquitin signals for the two most extensively studied linkages, K48 and K63, in vitro and in vivo. DARPins are high-affinity binders that can, in contrast to conventional antibodies, be used in vivo due to their small size and simple structure. They were raised against K48- and K63-linked ubiquitin dimers from a library with randomized target interaction residues by our collaboration partners from the Plückthun group (University of Zürich). A screen of all hits yielded a panel of highly selective DARPins, which were characterized with respect to their selectivity and inhibitory effects on cell growth and (de-)ubiquitylation. The most suitable candidate per linkage was further investigated and compared to the commercially available TUBE (tandem-ubiquitin binding entities) with regard to their specificity and affinity. Crystal structures revealed their binding modes, explaining their properties. The anti-K48 and anti-K63 DARPins are suitable for diverse pull-down applications and retain their selectivity in vivo as demonstrated by mass spectrometry and co-localization with respective antibodies. Tagged with a fluorophore, these DARPins can be used as biological sensors to specifically track K48- and K63-polyubiquitin chains in vivo, enabling the monitoring of the dynamics and localization of specific ubiquitin conjugates in various pathways, including their involvement in aggresome formation and the DNA damage response. A combination of both linkage-specific DARPins yielded a K48-K63-branched ubiquitin chain sensor, which demonstrated its specificity in vitro and showed potential as a biosensor for in vivo applications. The second part, entitled "Mechanistic analysis of the function of polyubiquitin chains in Exo1 recruitment for DNA damage bypass," endeavors to address the long-standing unresolved question of how the specific K63-linked ubiquitin chain on the Proliferating cell nuclear antigen (PCNA) initiates the DNA damage bypass pathway of template switching (TS). TS is the error-free pathway of the DNA damage bypass, which is critical to ensure complete and accurate duplication of the genome through DNA replication, thereby maintaining genome stability and avoiding potential carcinogenesis. TS and the K63-linked ubiquitylation as a starting signal have been known for approximately 20 years, yet no specific reader has been identified. We hypothesize that this reader could be the exonuclease Exo1, which has already been shown to be crucial for the TS pathway. Therefore, we investigated its direct interaction with PCNA and its ubiquitylated forms. A PCNA interaction motif in Exo1 was identified and characterized. We also set out to identify a ubiquitin binding domain by creating and analyzing the point mutants of a putative domain. Furthermore, the biological relevance of both motifs was studied to ascertain their involvement in the TS pathway.