Wilson, MDR; (2013) Proteasome-mediated processing of Def1: a critical step in the cellular response to DNA damage. Doctoral thesis (PhD), UCL (University College London).

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Abstract

DNA damage can pose an irreversible steric block to RNA polymerase II (RNAPII), preventing transcription. RNAPII becomes stalled at DNA lesions, blocking normal repair. As a ‘last-resort’ mechanism to clear the stalled polymerase - and repair the damage - the largest subunit of RNAPII is poly-ubiquitylated and degraded. In yeast, this process is dependent on the Def1 protein, through a previously unresolved mechanism. Using a combination of yeast genetic, biochemical and cell biological techniques this thesis reports the molecular mechanism of Def1 in this process. Upon DNA damage induced RNAPII stall, Def1 becomes ubiquitylated and partially proteolytically processed by the 26S proteasome. This creates a biologically active, shorter form of Def1, termed pr-Def1. Removal of the C-terminus of Def1, which usually promotes nuclear export, results in nuclear accumulation of the N-terminal processed fragment. Nuclear pr-Def1 binds to stalled, mono-ubiquitylated RNAPII and recruits the Elongin-Cullin ubiquitin ligase complex, promoting RNAPII polyubiquitylation and degradation. Interestingly, Def1’s ubiquitin-binding CUE domain and a novel ubiquitin homology domain in the Elongin complex mediate this interaction. These results outline the multi-step mechanism of RNAPII poly-ubiquitylation, elucidate Def1 activation and function, and identify an atypical ubiquitin-like domain in the yeast Elongin complex.

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