Fig. 5: DNA damage induced in S-phase by TRAIP degradation depends on presence of transcription machinery.
From: TRAIP resolves DNA replication-transcription conflicts during the S-phase of unperturbed cells
A HCT116 TRAIP-mAID cells were arrested in G1 stage of the cell cycle and TRAIP degraded before release of cells into S-phase. Upon S-phase entry cells were exposed to triptolide for 90 min. γH2AX and 53BP1 foci induced were visualised by immunofluorescence and quantified. Example pictures (left) and quantification of three independent experiments is presented as quantification of number of γH2AX and 53BP1 foci per nucleus (middle) or percentage of positive cells (>0 foci) in population (right) (mean ±SEM)). Differences between conditions was identified using t tests (yH2AX: TRAIP-mAID C2 - Triptolide: p = 0.00622; TRAIP-mAID D5 - Triptolide: p = 0.00798; TRAIP-mAID C2 + IAA ±Triptolide: p = 0.0297; TRAIP-mAID D5 + IAA ±Triptolide: p = 0.0318). 53BP1: TRAIP-mAID C2 - Triptolide: p = 0.0424; TRAIP-mAID D5 - Triptolide: p = 0.0474; TRAIP-mAID C2 + IAA ±Triptolide: p = 0.0286; TRAIP-mAID D5 + IAA ±Triptolide: p = 0.03709). B TRAIP was degraded in hTERT-RPE1 TRAIP-mAID cells for 24 h. In the last 90 min, cells were optionally exposed to triptolide and in the last 20 min, cells pulsed with EdU. γ−H2AX, 53BP1 foci, and EdU incorporation were visualised by immunofluorescence and quantified. Example pictures (left) and quantification of 2–3 independent experiments is presented as number of γ−H2AX and 53BP1 foci per EdU-positive nucleus. Mann–Whitney t test; ****p value < 0.0001. C HCT116 TRAIP-mAID cells were treated as in A but instead of DNA damage foci analysis, γH2AX ChIP was conducted followed by RT-PCR to detect indicated TSS. N2 is a amplifying a fragment from gene desert on chromosome 13. Mean of n ≥ 3 with SEM. T.test per gene per condition: PH4B ctr vs IAA p = 0.034, WDR ctr vs IAA p = 0.018, WDR IAA vs IAA+Trip p = 0.016. D TRAIP-mAID cells were arrested in G1 stage of the cell cycle and TRAIP degraded before release of cells into S-phase. In early S-phase EdU was added for 20 min and proximity between incorporated EdU and RNA Pol II visualised by PLA. Example pictures (top) and quantification all PLA events per nucleus are presented (bottom). Mann–Whitney t test; ****p value < 0.0001 (n = 3). E PLA between PCNA and RNA Pol II (left) and AND-1 and RNA Pol II (right) after optional treatment with auxin. Experiments were carried out analogously to that detailed above; cells were arrested in G1 where TRAIP was degraded through IAA treatment, before being released into S-phase, and samples taken ~12.5 hours post release. Example pictures (top) and quantification of all PLA events per nucleus are presented (bottom). Mann–Whitney t test; ****p value < 0.0001 (n = 3). Source data are provided as a Source Data file.
