Transcription-blocking lesions (TBLs) stall elongating RNA polymerase II (PolII), which then initiates transcription-coupled repair (TCR) to remove TBLs and allow transcription recovery. In the absence of TCR, eviction of lesion-stalled PolII is required for alternative pathways to address the damage, but the mechanism is unclear. This study, utilizing Protein-Associated DNA Damage Sequencing (PADD-seq), reveals that the p97-proteasome pathway can evict lesion-stalled PolII independently of repair. Both TCR and repair-independent eviction require CSA and ubiquitination. However, p97 is dispensable for TCR and PolII eviction in TCR-proficient cells, highlighting repair’s prioritization over repair-independent eviction. Moreover, ubiquitination of RPB1-K1268 is important for both pathways, with USP7’s deubiquitinase activity promoting TCR without abolishing repair-independent PolII release. In summary, this study elucidates the fate of lesion-stalled PolII, and may shed light on the molecular basis of genetic diseases caused by the defects of TCR genes.
