Extended Data Fig. 8: PZL-A stabilizes mutant POLγ interactions with the template during active DNA synthesis.
From: Small molecules restore mutant mitochondrial DNA polymerase activity
a, Formation of stalled elongation complexes was analysed by EMSA in the absence or presence of PZL-A. Complex formation was plotted against POLγ concentrations, and the binding curves were fitted to a quadratic equation for tight-binding to determine the dissociation constants (Kd). Presented data are the mean ± s.d. (n = 3 independent experiments). b, Summary of the determined dissociation constants, Kd (nM) from experiments in a. Presented data are the mean ± s.e.m. (n = 3 independent experiments). c, Schematic representation of the competition experiment used to evaluate DNA binding of idling POLγ. The indicated POLγ variants were incubated with a radiolabelled template at room temperature in the presence of dCTP and dGTP. A 50-fold excess of unlabelled competitor template was added, and the reactions were incubated at 37 °C for the indicated times. Samples were separated using 4% native-PAGE, and the fraction of the formed complex was quantified and plotted against time to determine the dissociation rate constant. Drawing by Jennifer Uhler (copyright holder). d, The percentage of labeled DNA bound to POLγ during idling was plotted versus time (minutes after addition of unlabeled DNA) and the data were fit to an exponential dissociation model to determine the dissociation rate constant, koff. The fit to the wild-type data (dashed line) was added to the mutant plots for comparison. Data points are mean ± s.d. (n = 3 independent experiments). e, Summary of the determined dissociation rate constants, koff (min−1) from experiments described in c,d. Presented data are the mean ± s.e.m. (n = 3 independent experiments). f, Quantification of the rolling circle assay in Fig. 3e. Data are presented as replication products relative to WT (15 min time point). The values from three timepoints (15, 30 and 60 min) were plotted for each mutant ± PZL-A as well as wild-type. Data presented are mean ± s.d., n = 3 (mutants) or n = 6 (wild-type) independent experiments. For gel source data (a, b, d, e), see Supplementary Figs. 14–16.
