Fig. 3

Free mtDNA ends in HEK 293 cells expressing mitoEagI or mitoPstI. a Cumulative relative frequencies (CRF) of mtDNA ends detected by ultra-deep sequencing of linker-ligated mtDNA 6 h after induction of mitoEagI expression. CRF values for both orientations of ends were combined into a single curve. MGME1 knockout (red) results in persistence of non-degraded ends. In POLG p.D274A knockin cells (green), over 80% of ends are detected within a distance of 600 base pairs from the cutting site. Functionally relevant sites associated with prominent clusters of ends are marked on the top (mTER, mitochondrial transcription termination site; oriL, replication origin for the light strand; [oriH], replication origin region for the heavy strand). b, c Relative frequencies of blunt mtDNA ends at the vicinity of cutting sites detected by ultra-deep mtDNA sequencing 6 h after induction of mitoEagI (b) and mitoPstI (c). Balk heights represent proportions of ends at specific nucleotide positions among all detected ends of the same orientation. Shadings indicate the retained part of mtDNA. Gray, control; red, MGME1 knockout in mitoEagI and MGME1 siRNA knockdown in mitoPstI. Blue shading indicates mitoEagI and mitoPstI recognition sites (schematically shown on the top). d, e Frequent mtDNA ends distal to the cutting sites as observed by ultra-deep mtDNA sequencing 6 h after induction of mitoEagI (d) or mitoPstI (e) in control cells. Shaded areas indicate the retained mtDNA fragment. Associated GC stretches are indicated by red shading (at least 3 consecutive Gs or Cs starting at less than 3 nucleotides difference from the end). Note that prominent ends are located at different sides of the same GC stretch depending on the main direction of degradation in mitoEagI-expressing and mitoPstI-expressing cells (indicated by arched arrows in the schemes on the side). The presence of non-degraded and selected partially degraded ends was confirmed by single-molecule PCR (Supplementary Fig. 5b)