Extended Data Fig. 8: rNTP/dNTP ratio influence rate of mtDNA synthesis.
From: Ribonucleotide incorporation into mitochondrial DNA drives inflammation
a, EdU labelling of Yme1l+/+ and Yme1l−/− MEFs for 4 h to visualize newly synthesized DNA (yellow), following by TOMM20 antibody staining (magenta). Confocal images are shown including an enlargement image of the inset. Nuclear genomic replication is shown together with smaller foci of newly synthesized mtDNA. Scale bars, 5 μm. Each quantified dot (right panel) represents the average number of newly synthesized mtDNA foci per cell in ten different images. b, mtDNA depletion/repopulation assay in Yme1l+/+ and Yme1l−/− MEFs. After mtDNA depletion with 2´,3´-dideoxycytidine (ddC; 40 µM) and removal of ddC, mtDNA replenishment was monitored. n = 3 biologically independent experiments. c, Effects of increasing concentration of GTP on in vitro DNA replication, which was performed for 60 min at 37 °C. DNA products with incorporated radioactive nucleotides were separated on a 0.8% alkaline agarose gel. Template (T); Products (P). d, DNA replication reactions containing recombinant POLγ, mtSSB, and a radiolabeled, primed single-stranded DNA template were incubated with increasing concentrations of CTP, UTP, or ATP (0, 10, 50, 100, 200, 300, 400, and 500 µM for each NTP) for 15 min at 37 °C. DNA synthesis products were separated on an agarose gel. Full-length (FL) product and the radioactively labelled primer/template (T) are indicated. e, Quantification of Southern blot analysis of alkaline agarose gel resolving mtDNA isolated from wild-type MEFs treated with DMSO (control), 5 µM 5-FU and 100 nM HU. Signal intensities of individual pixels were determined to generate plots for untreated (left panel) and RNaseH2-treated samples (right panel). The intensity data were scaled between 0 and 1. P values were calculated using unpaired two-tailed Student t-test (a) or using two-way ANOVA with Šidák’s multiple comparison test (b). Data are presented as mean ± SD.
