Figure 6.

Elevated Twinkle expression induces biphasic θ replication in mtDNA. 2D-AGE analysis of HEK293T cell DNA from cells expressing no transgene (control); transgenic wild-type Twinkle (Twk) or Twinkle D311A (Twk-mut2), both with HA tags. Whole cell DNA was digested with (A) Pvu2 that cuts mtDNA at a single site in the minor arc (nt 2650), or (B) Bgl1 (sites at np 6034 and 6964). SSN, single-strand-nuclease. Interpretations of the mtRIs are shown beneath the gel images, b – bubble structures, dY – double Y arc. The strong dY is indicative of slow-replication in the minor arc (as per the map to the left of B), mtDNA was detected with probe h1. Interpretations of the mtRIs are shown beneath the gel images, c – circles, b – bubble structures (l – large, s – small), dY – double Y arc, e – eyebrow (the red cross indicates a blocked site, which is attributable to RNA/DNA hybrid as the dY arc associated with Bgl1 digests that encompasses the minor arc is SSN-sensitive (Supplementary Figure S5A)). Some loss of RNA/DNA hybrid is expected during extraction because the source material was cultured cells and there was no purification of mitochondria, both of which result in RNA loss and the appearance of gaps that enable SSN to convert b and e forms to broken bubbles (bb), dY and X structures (termination intermediates), as illustrated beneath the gel images of panel A (see text and (14,51) for further details).