Fig. 1: Mutations impair polymerase activity across all tested dNTP concentrations.

a Schematic representation of the in vitro replication assay on a single-stranded template. A short 20-nt primer was annealed to circular, single-stranded M13mp18 DNA, providing a pre-existing 3’-OH group from which POLγ could initiate DNA synthesis and proceed until synthesizing the full molecule. b–d Both hPOLγWT and mPolγWT, as well as their mutant variants, were tested across a broad range of dNTP concentrations. Polymerase and human mtSSB were incubated with a primed, single-stranded DNA template at 37 °C for 20 min and analyzed on a 0.8% native agarose gel. The positions of the full-length (FL) product and the radiolabeled primer template (T) are indicated. All mutant variants exhibited reduced activity at all tested dNTP concentrations compared to their respective WT counterparts. n = 3 independent experiments. e Comparison of DNA synthesis activity between hPOLγ^WT and disease-causing mutations in hPOLγ. The assay was performed at lower Polymerase concentrations and with 10 µM dNTPs to ensure synthesis remained in the linear phase. DNA synthesis activity was assessed using the template described in (a) with an unlabeled primer. Polymerase activity was measured after 60 min at 37 °C by monitoring SYBR Green fluorescence, indicative of newly formed double-stranded DNA, enabling comparison between the variants. f Comparison of DNA synthesis activity between mPolγ variants and mPolγ^WT. The experimental conditions were similar to (e). Relative activity to corresponding wild type (Mean, %) indicated above the bar. The error bars present the standard deviation and n = 16 technical replicates in (e, f). For multiple comparisons in e and f, Welch and Brown-Forsythe one-way ANOVA was used to determine p values. ****p < 0.0001. Source data are provided as a Source Data file.