The influence of exonuclease activity on rNMP incorporation by Pol γ.

<p>(<b>A</b>) Schematic view of reaction set up in Fig 3B. (<b>B</b>) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo^-) Pol γ. <i>In vitro</i> replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α-³²P]-dCTP. Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. (<b>C</b>) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo^- (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. (<b>D</b>) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT <i>PolgA</i> (n = 2) and exonuclease-deficient <i>PolgA</i>^<i>D257A</i> (n = 3; “exo^-”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). (<b>E</b>) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT <i>PolgA</i> and <i>PolgA</i>^<i>D257A</i> mice.