RNA polymerase II is released from the DNA template during transcription-coupled repair in mammalian cells

Yi-Ying Chiou,Jinchuan Hu,Aziz Sancar,Christopher P Selby

doi:10.1074/jbc.ra117.000971

Abstract

In mammalian cells, bulky DNA adducts located in the template but not the coding strand of genes block elongation by RNA polymerase II (RNAPII). The blocked RNAPII targets these transcription-blocking adducts to undergo more rapid excision repair than adducts located elsewhere in the genome. In excision repair, coupled incisions are made in the damaged DNA strand on both sides of the adduct. The fate of RNAPII in the course of this transcription-coupled repair (TCR) pathway is unclear. To address the fate of RNAPII, we used methods that control transcription to initiate a discrete "wave" of elongation complexes. Analyzing genome-wide transcription and repair by next-generation sequencing, we identified locations of elongation complexes and transcription-repair coupling events in genes throughout the genome. Using UV-exposed human skin fibroblasts, we found that, at the dose used, a single wave of elongation complexes was blocked within the first 25 kb of genes. TCR occurred where the elongation complexes were blocked, and repair was associated with the dissociation of these complexes. These results indicate that individual elongation complexes do not engage in multiple rounds of TCR with successive lesions. Our results are consistent with a model in which RNAPII is dissociated after the dual incision of the transcription-blocking lesion, perhaps by Cockayne syndrome group B translocase, or during the synthesis of a repair patch.

Highlights

In mammalian cells, bulky DNA adducts located in the template but not the coding strand of genes block elongation by RNA polymerase II (RNAPII)
The DRB effect is reversible, and to create a discrete “wave” of RNAPs transcribing along the template, we used a pulse– chase–pulse scheme termed double DRB (DRB2) treatment illustrated in Fig. 1A, in which cells were first incubated in DRB for 2 h, to prevent new, actively elongating complexes and to permit RNAPII complexes already elongating during treatment with DRB to complete and terminate transcription
After 2 h, which is sufficient for the completion of even the longest transcripts, the DRB was washed off, and the cells were incubated without DRB for 10 min

Summary

Control of transcription with DRB

Our experiments utilized the transcription inhibitor 5,6dichlorobenzimidazole 1-␤-D-ribofuranoside (DRB) to control RNAPII elongation. The DRB effect is reversible, and to create a discrete “wave” of RNAPs transcribing along the template, we used a pulse– chase–pulse scheme termed double DRB (DRB2) treatment illustrated, in which cells were first incubated in DRB for 2 h, to prevent new, actively elongating complexes and to permit RNAPII complexes already elongating during treatment with DRB to complete and terminate transcription. During the 10-min DRB-free period, RNAPII elongation begins from promoter proximal pause sites in a synchronized manner. The increase in progressively distal RNA levels with time illustrates a discrete wave of RNAPII transcribing along the gene at ϳ1 kb/min during the DRB2 treatment, in agreement with reported rates of RNAPII elongation [24]

Overall effects of controlled transcription on repair

Global analysis of individual elongation complexes and repair events

Analysis of a discrete wave of repair

Analysis of a discrete wave of transcription

Discussion

Cells culture and DRB treatment