Purification of Mitochondrial DNA Polymerase

Pol γA and Pol γB purification was carried out following the previously published protocol (57 (link)). Briefly, PolγB ΔI4 variant was expressed in E. coli BL21-RIL and purified using Ni-NTA agarose (Qiagen) and Mono S affinity chromatography. Pol γA was expressed in Sf9 cells and purified on TALON (Clontech) and Superdex200 size exclusion columns. Purified Pol γA was mixed with Pol γB at a 1:2 M ratio and applied to the Superdex200 gel filtration column. Peak fractions corresponding to the Pol γAB holoenzyme heterotrimer were pooled and concentrated. Purity was judged to be ∼98% using SDS-PAGE. PARP-1 was expressed in E. coli BL21 (DE3) and purified per a published protocol (58 (link)). Following cell lysis, PARP-1 was purified by sequential application to Ni-NTA agarose (Qiagen), HiTrap Heparin HP column (GE Healthcare), and Superdex200 chromatography columns. Human Poly(ADP-ribose) glycohydrolase catalytic domain (deletion of N-terminal 455 amino acids) (hPARG-ΔN455) was purified according to Tucker et al. (59 ). After expression in E. coli BL21 (DE3), hPARG-ΔN455 was purified by sequential application to Ni-NTA agarose (Qiagen) and Superdex200 chromatography columns.