Molecular activation mechanism of RNA polymerase II degradation factor Def1

Transcribing RNA polymerase II (RNAPII) is prone to stalling and arrest due to its inability to efficiently bypass DNA lesions. RNAPII stalled at a DNA lesion triggers the transcription-coupled nucleotide excision repair (TC-NER) pathway to immediately repair the lesion and allow transcription to progress. However, if the TC-NER pathway fails, the so-called ‘last resort pathway’ is activated. The last resort pathway leads to the removal of stalled RNAPII from DNA, clearing the way for other repair factors. Ubiquitin signalling plays a crucial role in this pathway. The last resort pathway is comprised of several ubiquitin-dependent steps. Rpb1, the largest subunit of RNAPII, undergoes two sequential ubiquitylation events: first, mono-ubiquitylation is carried out by ubiquitin ligase Rsp5, and then polyubiquitylation by the Elongin-Cullin complex. These steps enable the ubiquitindependent chaperone Cdc48, together with its adaptor proteins Ubx4 and Ubx5, to disassemble RNAPII from chromatin, and present it to the proteasome. Importantly, for the second ubiquitylation step to occur, RNAPII degradation factor Def1 undergoes ubiquitin- and proteasome-dependent activation, which allows it to bind RNAPII and recruit the Elongin-Cullin complex. This thesis focuses on the molecular detail of Def1 activation in response to DNA damage in Saccharomyces cerevisiae