Gaul, Liam; (2021) Characterisation of CSB ubiquitylation in response to UV-induced DNA damage. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A variety of DNA repair pathways operate in different cellular contexts to tackle a diversity of DNA lesions and maintain genome stability. Nucleotide excision repair (NER) recognises and removes a variety of helix-distorting lesions, operating via two pathways: a global-genome (GG-NER) and transcription-coupled (TC-NER) pathway. GG-NER repairs damage at any locus in the genome thus promoting genome stability by abrogating replication-mediated stress and mutagenesis. TCNER on the other hand is restricted to the template strand of actively transcribed genes and provides means to rapidly repair lesions that would otherwise impair transcription. Thus, TC-NER has seemingly evolved to not only maintain genome stability but sustain transcription by removal of RNA polymerase II (RNAPII)-stalling lesions. The two pathways differ in their mode of recognition of lesions while downstream repair steps of excision and DNA synthesis are mutual. GG-NER relies on XPC and UV-DDB that recognise and bind directly to lesions, initiating repair. TC-NER is initiated by a lesion-stalled RNAPII, which is recognised by Cockayne’s syndrome B (CSB) prompting recruitment of further repair factors. Mutations in CSB or CSA result in Cockayne’s syndrome, a disease characterised by photosensitivity, neurological deficiencies and progeria. UV-DDB and CSA reside in ubiquitin ligase complexes highlighting the importance of ubiquitylation in NER. The Svejstrup laboratory previously identified a ubiquitinbinding domain in CSB that was essential for its function as well as several CSAand UV-dependent ubiquitylation sites on CSB. Building on this work, I have developed a cell system exclusively expressing CSB mutants that are not ubiquitylated in response to UV-irradiation. I present evidence that ubiquitylation of CSB is necessary for the recovery of transcription and cell survival following UVirradiation. Inhibition of CSB ubiquitylation does not affect its recruitment to chromatin following UV, indicating it is a step downstream of the recognition and binding of a stalled RNAPII. These data support the hypothesis that CSB ubiquitylation is a vital step in TC-NER.

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