Extended Data Fig. 7: Local transcription enables NER.

Supplementary to Fig. 5. a–d, Depriving the genomic loci of transcription abolishes their NER. a, Schematics of the mCherry insulators. A transcription unit containing mCherry (with or without lacZ promoter) flanked by the terminator cassettes was inserted at the tam and nupG loci. b–d The expression of mCherry from the insulators (b) and RNAP occupancy (c, d) after IPTG induction, as determined by RT-qPCR and ChIP–qPCR, respectively. Values are the mean ± SD from 3 independent experiments. e, f, CPD repair within the insulators. Most of NER strictly depends on promoter-initiated transcription. The levels of transcription and NER are stronger within the nupG insulator comparing to the tam insulator. Cells were induced with IPTG followed by UV irradiation (40 J/m²) and recovery in the dark for the indicated time intervals. CPD density was determined by SLR-qPCR as in Fig. 5a and used to calculate the percentage of repaired CPDs. Values are the mean ± SD from 3 independent experiments. g–j, UvrAB recruitment to the UV-damaged DNA strictly depends on local transcription in both tam and nupG loci. Occupancy of RNAP (c, d), UvrA (g, i) and UvrB (h, j) following UV irradiation was determined by ChIP–qPCR. Cells were induced with IPTG followed by UV irradiation (40 J/m²) and recovery for the indicated time intervals. Values are the mean ± SD from 3 independent experiments. Results are shown as a fold change in the occupancy of UvrAB within the insulator following UV irradiation. UT – untreated. Values are mean ± SD from 3 independent experiments. **P < 0.01, ****P < 0.0001 (Statistical analysis was performed using unpaired non-parametric two-tailed Mann-Whitney t test). P values compare the percentage of DNA repair between “promoter” and “no promoter” strains for a given time point.