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Abstract

DNA damage response (DDR) is a well-characterised process, however, the majority of studies so far focus specifically on nuclear events, while the cytoplasmic and endomembrane response to DNA damage remains largely unexplored. Although to date there exists little experimental work providing evidence to suggest a role of the Golgi complex as part of the cellular DNA damage response (DDR), a few and far in-between studies have alluded to this function (Farber-Katz et al., 2014). In line with this, previous work from our lab identified a network of 15 proteins that function in various distinct DNA repair pathways, along with cell cycle and other regulatory proteins, that localise to the Golgi and the nucleus. A first in-depth characterisation of the dual-localising DDR protein RAD51C has shed some light on the potential role the Golgi complex might play in DDR (Galea et al., 2022). This work has revealed that the Golgi localisation of RAD51C is dependent on the Golgin Giantin, and strongly reduced upon DNA damage coincident with its increase in nuclear DNA damage sites. The loss of this Giantin-RAD51C interaction by siRNA-mediated depletion of Giantin leads to genomic instability and aberrant DNA repair. In light of these findings and the wide array of DDR proteins identified at the Golgi, I hypothesised that similar regulatory mechanisms would be present for various other DNA repair pathways. To assess the role of the DDR Golgi population in DNA repair response, various types of DNA lesions were induced utilising DNA damaging agents, while monitoring for any change in distribution patterns of the investigated DDR proteins. The dual-localised DDR proteins were found to change in localisation in a bi-directional manner, showing distribution from the Golgi to the nucleus or from the nucleus to the Golgi depending on the DNA damage induced and based on the proteins’ role in specific DDR pathways. A sub-organelle distribution analysis of DDR proteins within the Golgi was then performed, speculating that specific sub-organelle distribution could be important for the activation and regulation of these proteins at the organelle. The dual-localised DDR proteins were found to be heterogeneously distributed throughout the Golgi stacks and their position on the organelle was found to be correlated to their function in DDR. Additionally, this work identified several Golgins acting as anchors and regulators of these DDR proteins at the Golgi. Furthermore, proteomic-scale interaction analysis of DDR and Golgin family proteins revealed the new interactions between the Golgin and DDR proteomes, suggesting that the network of interactions between the Golgi proteins and DDR proteins might be more extensive than previously found. Altogether, the results described in this work demonstrate the presence of a complex relationship between the Golgi and DDR, identifying new interacting pathways between the Golgi proteins, in particular Golgins and DDR. These findings expand on our current knowledge of how DDR is regulated, showcasing the Golgi as a regulatory hub for DNA damage response. These findings are a beginning of a new direction in the DNA damage repair field, shifting from a nuclear-centric to a more global picture of how DNA damage response is regulated, opening up possibilities for finding new therapeutic targets.