Extended Data Fig. 1: Effects of PZL-A on mutant POLγ activities.
From: Small molecules restore mutant mitochondrial DNA polymerase activity
a, PZL-A (1 μM) stimulates DNA synthesis in indicated mutant POLγ variants across a broad range of dNTP concentrations. Mutant POLγ variants and mtSSB were incubated with a primed, single-stranded DNA template at 37 °C. Samples were taken at the indicated time points and analyzed on a 0.8% native agarose gel. The positions of the full-length (FL) product and the radiolabelled primer template (T) are indicated. Primers may dissociate from the circular ssDNA during electrophoresis, explaining the lower signals in the template control lanes (-). Representative gels are shown (n = 2 independent experiments). b, EMSA (electrophoretic mobility shift assay) was used to determine the concentration of active POLγ holoenzyme–DNA complex (active enzyme) under the conditions used in the kinetic experiments (Fig. 1d). The fraction of template-bound POLγ holoenzyme was calculated relative to the unbound template. The concentration was calculated by multiplying the fraction active enzyme by 0.5 nM (maximum theoretical enzyme–DNA complex concentration). Data are presented as the mean ± s.d. (n = 3 independent experiments). c, Schematic representation of the exonuclease assay used to investigate effects of PZL-A on the exonuclease activity of POLγ. Drawing by Jennifer Uhler (copyright holder). d, Time-course experiments reveal that wild-type POLγ efficiently removes a mismatch at the 3′-end, whereas an exonuclease-deficient, mutant POLγ variant (D274A) is unable to remove the mismatch. POLγ was incubated with the mismatch-template at 37 °C in the absence of dNTPs for times indicated. The products were separated on a 10% urea-PAGE sequencing gel. The positions of products (P) and the radiolabelled mismatch template (T) are indicated. The fraction of short oligonucleotides (shorter than the 19-mer) was quantified and plotted versus time to visualize the DNA degradation. Data are presented as the mean ± s.d. (n = 3 independent experiments). e–f, Time-course experiments demonstrate that PZL-A (1 μM) has no observable negative effect on the exonuclease activity of mutant POLγ variants tested. The experiments were performed as in d. Data are presented as the mean ± s.d. (n = 3 independent experiments). For gel source data (a, b, d–f), see Supplementary Figs. 9–13.
