Targeted Protein Degradation

Helix distorting DNA damage, that is encountered by RNAPII, is mostly repaired by transcription-coupled nucleotide excision repair (TC-NER). When TC-NER cannot allow for continued transcription, the stalled RNAPII is removed to allow for access by DNA repair proteins that function to repair the DNA lesion. Removal is thought to be achieved by poly-ubiquitination and degradation of RPB1, the largest subunit of RNAPII. Hence, degradation of RPB1 following DNA damage functions to regulate two fundamental cellular process: DNA repair and transcription.

Our overall goal is to understand how the UPS interacts with RNAPII to clear stalled transcription complexes from sites of DNA damage, in human cells. Firstly, we will identify genetic factors required for (de)stabilization of RPB1. To do so we will use a focused sgRNA CRISPR library targeting E3 proteins, deubiquitylating (DUB) enzymes, and accessory proteins (that function within this pathway), hence identifing which proteins (de)stabilize RPB1. Secondly, we will identify potential E3 ligases and DUB enzymes that add or remove poly-ubiquitin from RPB1, taking orthogonal proteomics-based approaches. Finally, we will investigate the mechanisms by which the identified E3s and DUBs affect RPB1 degradation and stabilization, as well as the subsequent cellular outcomes. Here, we will map the type and position of ubiquitylation mediated by the identified E3 and removed by the DUB. We will determine the impact of ubiquitylation/deubiquitylation of RPB1 on DNA repair and transcription.

Taken together, this multidisciplinary proposal will clarify mechanisms and outcomes of RNAPII degradation following DNA damage, in order to coordinate transcription and DNA repair.