RNA polymerase II (RNAPII) transcription is a highly regulated and essential process. The carboxy-terminal domain (CTD) of human RNAPII consists of 52 repeats of the consensus heptad Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, which undergo dynamic, regulatory phosphorylations. Phosphorylation of CTD S2/S5 is well-characterised hallmark of active transcription of protein-coding genes; CTD Y1 phosphorylation (CTD Y1P) is less understood. CTD Y1P functions as anti-termination signal by blocking recruitment of termination factors to actively transcribing RNAPII in S. cerevisiae. In mammals, CTD Y1P is enriched at promoters and associated with antisense transcription. Transcription is generally regarded as a threat for genome stability and globally impaired by physical blockage, concomitant with heterochromatin formation during DNA repair. However, global analysis of nascent RNA levels identified a subset of damage-induced short, non-coding transcripts, preceding inhibition of RNAPII elongation. Intriguingly, DSBs are repaired faster, when induced in actively transcribed loci.
To investigate RNAPII in response to DNA damage, we employed a U2OS cell line harboring the 4-Hydroxytamoxifen (4OHT)-inducible endonuclease AsiSI-ER (AsiSI-ER U2OS), which allows localised sequence-specific induction of DSBs and new generation sequencing methods.