Fig. 3: PZL-A increases processivity of mutant POLγ and restores mitochondrial replisome function.

a, Mutant POLγ variants and mtSSB were incubated with a primed, single-stranded DNA (ssDNA) template at 37 °C. Samples were taken at the indicated time points and analysed 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 = 3 independent experiments). b, Schematic representation of the processivity assay. Heparin is used to trap free POLγ, preventing dissociated enzyme from rebinding to the template to continue DNA synthesis. c, Mutant forms of POLγ have impaired processivity compared with wild-type. Processivity is restored in the presence of PZL-A. The products were separated on an 8% urea-PAGE sequencing gel. The positions of products (P) and the radiolabelled template (T) are indicated. Representative gels are shown (n = 3 independent experiments). d, Schematic representation of the rolling-circle assay used to investigate the effects of PZL-A on replisome function on a dsDNA template. e, Time-course experiments as outlined in d demonstrate that PZL-A activates mutant POLγ variants during Twinkle DNA helicase-dependent rolling-circle DNA replication. DNA synthesis was monitored by the incorporation of radiolabelled nucleotides and the resulting products of increasing length were separated on a 0.8% alkaline agarose gel (quantified in Extended Data Fig. 8f). Representative gels are shown (n = 3 independent experiments). For source data of gels in a,c,e, see Supplementary Figs. 4–7. Drawings in b,d by Jennifer Uhler (copyright holder).