rNMP incorporation frequency of Pol γ and the mtDNA replisome on long DNA templates.

<p>(<b>A</b>) Schematic diagram of the 7.3 kb M13 ssDNA substrate used to compare the incorporation of rNMPs by Pol γ and yeast Pol δ in the primer extension assay in Fig 2B. The newly synthesized DNA was labelled by addition of [α-³²P]-dCTP to the reaction. (<b>B</b>) Analysis of the rNMP incorporation frequency of Pol γ and yeast Pol δ at “normal” (“N”, S phase) concentrations, “low” (“L”, concentration during the rest of the cell cycle and in non-dividing cells), or <i>S</i>. <i>cerevisiae</i> (“Sc”) dNTP concentrations, in the absence or presence of rNTPs. In all reactions, the DNA template was coated with the relevant single-stranded DNA-binding proteins (lanes 1–6 with RPA; lanes 7–14 with mtSSB) to avoid stalling of DNA synthesis due to formation of DNA secondary structures. Untreated (“-NaOH”) or alkaline (“+NaOH”) treated reaction products were analysed on an agarose gel under denaturing conditions. To estimate rNMP incorporation frequencies from the presented gel, the median length of alkali stable products was determined and used to calculate the frequencies as described in Materials and Methods. Numbers on the left-hand side of the gel indicate positions of DNA marker bands and the full-length starting product (7.3) in kb. The gel is a representative picture of four independent experiments. (<b>C</b>) Schematic diagram of the primed mini-circle substrate with a 40 nt 5′ overhang used in Fig 2D. (<b>D</b>) Analysis of the rNMP incorporation frequency by the mitochondrial replisome consisting of mtSSB, Twinkle and Pol γ (AB₂) on a primed mini-circle substrate with a 5′ overhang for Twinkle loading. Reactions were carried out at normal (“N), low (“L”), and “<i>S</i>. <i>cerevisiae</i>” (“Sc”) dNTP concentrations in the presence or absence of rNTPs. Untreated (“-NaOH”) and alkaline (“+NaOH”) treated reaction products were analysed on a denaturing (alkaline) agarose gel. The gel is a representative picture of two independent experiments.