Fig. 4

GC stretches decelerate mtDNA degradation. Relative frequencies of nucleotides at the vicinity of detected blunt double-stranded mtDNA ends are shown. Nucleotides surrounding all ends detected by ultra-deep sequencing of linker-ligated mtDNA were counted and their relative frequencies were normalized to overall nucleotide frequencies in the mitochondrial genome. Ends at the immediate vicinity of the cutting site were excluded from the analysis (mitoEagI: positions 2552–2585; mitoPstI: positions 6810–7015 and 8920–9125). To avoid bias through the most prominent ends in mitoEagI cells, corresponding positions were also excluded (positions 5732–5742 and 3208–3215). ‘dox+’, samples taken 6 h of inducing mitoEagI or mitoPstI expression. ‘dox−’, no induction, only leaky mitoEagI or mitoPstI expression. Upper set of panels shows ends generated by degradation in the forward direction, lower set of panels in reverse direction (according to reference numbering). Faded balks represent removed nucleotides. a Note that the high frequency of guanine and cytosine residues at the first 6 positions of linear mtDNA species upon induced cleavage in control mitoEagI cells. b Similar pattern can be observed in mitoPstI cells, although, cutting sites localize to different parts of the mitochondrial genome. c MGME1 knockout cells and d POLG p.D274A knockin cells do not show differences between induced and non-induced conditions. This is in line with the observation that newly generated ends do not undergo rapid degradation in the absence of MGME1 exonuclease or in the presence of exonuclease-deficient POLG