Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic because of its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we have taken advantage of a cell model with two different types of mitochondrial genomes and impaired ability to turn over broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot. Combining our observations with previously published work, we propose that the mitochondrial replisome can catalyze microhomology-mediated recombination of linear mtDNA ends, thus rendering a specialized mitochondrial recombinase unnecessary. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

The data consists of original FASTQ files of nanopore sequencing data. 

Mitochondrial DNA was linearised through the digestion of isolated mitochondrial with BamHI at 37°C for 2 h. Size selection was done using AMPure XP beads with a bead-to-volume ratio of 0.8. Nanopore sequencing was performed in accordance with the Native barcoding genomic DNA protocol with EXP-NBD104, EXP-NBD114 and SQK-LSK109 (version NBE_9Q65_V109_revAE_14Aug2019).

The end processing was done according to the manufacturer’s instructions using NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End repair/dA-tailing Module reagents. Barcode labelling was done with Native Barcoding Expansion 1-12 and NEB Blunt/TA Ligase Master Mix (M0367). Nanopore sequencing libraries were then prepared using 100 ng of linear mtDNA following the protocol of the ligation sequencing kit (Oxford Nanopore Technologies item number: SQK-LSK109). Adapter ligation was done with NEBNext Quick Ligation Reaction Buffer (NEB B6058), Adapter Mix II (AMII), and Quick T4 DNA ligase. The Nanopore sequencing libraries were then sequenced on the Oxford Nanopore Flongle device, using Flongle Sequencing Expansion (EXP-FSE001) and Flush Tether. Basecalling of the Nanopore sequencing was performed on the CSC Puhti system using Guppy v. 5.0.7.