Figure 4.

Elevated expression of wild-type or mutant Twinkle increases the abundance of fully-duplex DNA replication intermediates across the major arc of mtDNA. Mitochondrial DNA was analyzed by 2D-AGE from control HEK293T cells, or cells induced to express HA-tagged wild-type or mutant (D311A) Twinkle, after digestion with 1) Dra1, 2) BsaKHA1 or 3) Hpa1. Individual fragments were detected via hybridization to probes h2, h3 or h4 (see schematic map and Supplementary data). Y - replication fork arc, 1n – unit length fragment, 2n - twice the unit length fragment where the Y arc terminates. Standard Y arcs do not distinguish the direction of replication fork movement, and so forks could be entering from either end of all the fragments shown, as illustrated to the right of the map for the Dra1 fragment.