K63-linked ubiquitylation of S2P–RNAPII regulates transcription in a DNAPK inter-dependent manner in response to double-strand breaks

DNA double-strand breaks (DSBs) are highly toxic DNA lesions that can lead to genomic instability. DSBs can also interfere with other DNA-based processes, including transcription, and thereby jeopardizing cellular function. In situations of persistent DSBs, RNA polymerase II (RNAPII) needs to be removed to facilitate DNA repair. DSB-induced RNAPII removal involves multifaceted ubiquitylation, but the mechanisms involved remain elusive. Our data show that in response to DSBs, the E3 ubiquitin ligase NEDD4, and to a lesser extent CRL3 complexes, catalyse the ubiquitylation of elongating RNAPII, facilitating efficient DSB repair. Specifically, NEDD4 is identified as the specific writer of K63-linked ubiquitin chains on Serine2 phosphorylated (S2P)–RNAPII under stress, while the total pool of RNAPII is found to be modified mainly with K48-linked ubiquitin chains. We find that the ubiquitin ligases NEDD4, WWP2, and CUL3-based complexes exhibit a DNAPK inter-dependency, driving NHEJ repair and proper resolution of transcription defects caused by DSBs.