Fig. 5

Rearranged mtDNA molecules in HEK 293 cells expressing mitoEagI or mitoPstI. a Positions of mtDNA breakpoints as detected by ultra-deep sequencing of mtDNA in control cells 6 h after induction of mitoEagI. Continuous lines represent retained parts of the mitochondrial genome. Dotted lines indicate deleted regions in rearranged mtDNA species. Note that the observed breakpoints correspond to frequent ends (cf. Figure 3d). b PCR detection of breakpoints corresponding to non-degraded and partially degraded ends of the 2.1-kb mtDNA fragment in mitoPstI-expressing cells. Amplification primers MT8282F and MT7682R (Supplementary Table 4) are shown in the scheme as arrowheads. Note that the majority of detected breakpoints correspond to partially degraded mtDNA ends in control cells, while MGME1, POLG, or TWNK siRNA treatments result in breakpoints mainly corresponding to non-degraded mtDNA ends. The exact positions of representative breakpoints determined by single-molecule PCR and sequencing are shown in Supplementary Table 2. c Concatemers of the 2.1-kb mtDNA fragment in mitoPstI-expressing cells as detected by long-extension PCR⁸ using primers MT8194F and MT8387R (Supplementary Table 4). The shortest bands represent amplification products from unique copies of the 2.1-kb mtDNA fragment (lower arrowhead). Longer PCR products indicate the presence of multimers of the mtDNA fragment corresponding to non-degraded (upper arrowhead) or partially degraded (middle part) ends. Knockdown of the LIG3 gene decreases the abundance of concatemer species