Extended Data Fig. 6: PDHE1α PARylation is required for chromatin accessibility after DNA damage.

a, PDHE1α-deficient HeLa cells were reconstituted with PAR-binding site mutated or deleted constructs, and the cells were irradiated with 5 Gy IR and harvested for PDHE1α enzyme activity assay using a commercial assay kit. n = 3 biological replicates were collected. The statistical analyses were performed using a two-tailed student’s t-test, and data shows the mean ± s.d. b, IF staining showing the PARylated PDHE1α is required for pan-Ac signal accumulation on DSB sites. HeLa-PDHE1α-KO cells rescued with NLS-GFP-fused WT and PARylation-defective PDHE1α constructs were laser micro-irradiated and immuno-labeled with γH2AX and pan-Ac antibodies; nuclei were counterstained with DAPI. The pan-Ac intensity at DSBs was quantified (n >30). Scale bars, 5 μm. c, Quantification of γH2AX foci to confirm that similar amounts of DSBs were induced in the WT and all PDHE1α-mutant groups. The data representative of three independent replicates where data were collected from 100 cells per condition. d, MNase sensitivity assay to evaluate chromatin condensation. WT and PARylation-defective-construct-overexpressing PDHE1α-KO cells were treated with or without etoposide for 1 h. The gel image was captured by BioRad ChemiDoc XRS⁺, and the intensity of each nucleosome was calculated by Image J. e, HeLa cells and PDHE1α-KO cells were irradiated with 3 Gy IR followed by recovery for 1 h. The cellular chromatin fractions were extracted and analyzed by western blotting using antibodies against the indicated proteins. f, Accumulation of RFP-tagged 53BP1 and RPA70 at damage stripes on PDHE1α-deficient cells monitored by laser micro-irradiation-coupled live-cell imaging. RFP signal accumulation intensity at DSB stripes was quantified from three independent experiments (n = 20 cells were collected each group), the curve shows the mean ± s.e.m. Scale bars, 5 μm.

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