Fig. 1

Degradation of linearized mtDNA in control and mutant mitoEagI-expressing HEK 293 cells. a Southern blot showing the degradation of mtDNA within the first 18 h of induced expression of mitoEagI (E) in control, MGME1 p.I9Qfs*32 knockout (‘MGME1 ko’), and exonuclease-deficient POLG (‘POLG p.D274A’) cells. BamHI endonuclease-linearized DNA (B) was labeled with a mitochondrial probe represented by an asterisk as well as a probe specific for nuclear 18S ribosomal DNA (‘18S’). Note that persistent bands with one end in the vicinity of oriL in MGME1 ko and mutated POLG cells are already present before induction (time point ‘0’, lowest arrowhead). These linear mtDNA species are due to leaky mitoEagI expression and their presence is not related to the induced massive double-strand breaks. b Quantification of full-length mtDNA confirms the efficient cleavage of mtDNA by mitoEagI and (c) the persistence of mitoEagI-linearized mtDNA in MGME1 ko and POLG p.D274A cells. Band intensities were first normalized to 18S ribosomal DNA intensities then to intensities of the full-length mtDNA in each cell line before induction. Error bars represent standard errors of the mean (SEM) in three independent experiments (including both available MGME1 knockout clones). Significance was calculated by applying one-way ANOVA test. *P < 0.05, **P < 0.01, *** P < 0.001. d, e Coverage ratios throughout the mitochondrial genome as determined by ultra-deep sequencing of mtDNA from cells 6 h after induced mitoEagI expression and normalized to values in non-induced cells. In control (gray), coverage ratio is the lowest around the mitoEagI cutting site (represented by the two ends of the x-axis) and gradually increases in both directions before reaching full coverage. In MGME1 ko cells (d, red) and in cells with exonuclease-deficient POLG (e, green), coverage ratio drops only in the immediate vicinity of the mitoEagI cutting site. Note that library preparation techniques used for ultra-deep sequencing result in underrepresented positions in the close vicinity of free DNA ends