Investigating the cellular role of the deubiquitylating enzyme ZUP1, a novel player in the DNA damage response

Thesis

The DNA damage response (DDR) is a network of interrelated signal transduction pathways that identify and repair DNA lesions to maintain genome integrity. To coordinate the DDR, cells use post-translational modifications (PTMs) such as ubiquitin to modify and control protein fate and function. Importantly, ubiquitin signalling is a dynamic modification that can be edited or removed from substrates by deubiquitylating enzymes (DUBs). ZUP1 was recently discovered as the founding member of a new DUB class with selectivity against long K63-linked ubiquitin chains and a putative, but poorly defined, role in the DDR. This thesis aimed to improve our understanding of the cellular role of ZUP1 and define which DDR pathway(s) it functions in. To this end, I generated monoclonal ZUP1 knockout (KO) cell lines using CRISPR-Cas9 gene editing and found that these cells were sensitive to genotoxins that induce replication-coupled DNA damage, with hypersensitivity to the interstrand crosslinking agent mitomycin C (MMC), and the PARP inhibitors olaparib and talazoparib. Using the high-content immunofluorescence microscopy technique of quantitative image-based cytometry (QIBC), I then analysed the dynamics of key factors at stressed DNA replication forks and demonstrated that excess generation of ssDNA and persistence of RPA and RAD51 foci underlie the sensitivity of ZUP1 KO cells to MMC. Lastly, I investigated the chemogenetic interactors of ZUP1 in response to MMC in an unbiased manner using genome-wide CRISPR-Cas9 screening and identified potential synergistic interactions between ZUP1 and the factors BRCA2 and HELQ. Collectively, my results suggest that ZUP1 is a novel player in the response to replication-coupled DNA damage and provides new insight into its cellular roles.

Authors: Drake, M

• DOI: 10.5287/ora-kzv4ypqby
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Genome-wide CRISPR screen; Interstrand crosslink repair; DNA damage response; Deubiquitylase; DNA repair
• Subjects: Biochemistry; Cell Biology; DNA repair