UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-d amaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptio nally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DN A damage induction. Here, we investigated the effect of UV irradiation on t ranscription by using an in vitro transcription system that allowed uncoupl ing of initiation from elongation events. Nuclear extracts prepared from UV -irradiated or mock-treated normal human and CS cells were assayed for tran scription activity on an undamaged P-globin template. Transcription activit y in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a s trongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced tran scription activity in extracts coincided with a strong reduction of RNA pol ymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the fo rm of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely b y blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of tr anscription. CS proteins may be required for this process in a yet unknown way.