Fig. 4: NAD⁺ stimulates PARP1-mediated PARylation of mitochondrial DNA.

ADPr-ChAP was performed on purified mitochondria from WT (a) and PARP1-KO (b) cells that had been treated with 500 µM NAD⁺ with/without 5 µM olaparib for 30 min at 37 °C. After the reactions, mitochondrial samples were fixed for ADPr-ChAP. ADPr-ChAP purified mitochondrial DNA was subjected to real-time PCR using the indicated mtDNA-specific primers. Error bars represent the standard deviation. c ADPr-ChAP purified DNA from WT and PARP1-KO cells was sequenced, and the peak distribution on mtDNA was plotted by Integrative Genomics Viewer (IGV). d ADPr-ChIP sequencing reads of WT and PARP1-KO cells were aligned with seqMINER (Ver 1.3.3) on NC_012920 with 37 mitochondrial gene start sites as reference coordinates. Signal intensities are shown in the range of the mitochondrial gene start site ± 500 bp. C, control; N, + NAD; O, + NAD + olaparib. e, f Merged dataset profiles of aligned sequencing reads in D. One-way ANOVA was used to analyze the 7 groups followed by post hoc multiple pairwise comparisons using FDR correction to calculate p values for the pairwise comparisons. Error bars represent the standard deviation. *, <0.05; **, <0.01; ***, <0.001.