Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks

Abstract

To investigate mtDNA recombination induced by multiple double strand breaks (DSBs) we used a mitochondria-targeted form of the ScaI restriction endonuclease to introduce DSBs in heteroplasmic mice and cells in which we were able to utilize haplotype differences to trace the origin of recombined molecules. ScaI cleaves multiple sites in each haplotype of the heteroplasmic mice (five in NZB and three in BALB mtDNA) and prolonged expression causes severe mtDNA depletion. After a short pulse of restriction enzyme expression followed by a long period of recovery, mitochondrial genomes with large deletions were detected by PCR. Curiously, we found that some ScaI sites were more commonly involved in recombined molecules than others. In intra-molecular recombination events, deletion breakpoints were close to or upstream of ScaI cleavage sites, confirming the recombinogenic character of DSBs in mtDNA. A region adjacent to the D-loop was preferentially involved in recombination of all molecules. Sequencing through NZB and BALB haplotype markers in recombined molecules enabled us to show that in addition to intra-molecular mtDNA recombination, rare inter-molecular mtDNA recombination events can also occur. This study underscores the role of DSBs in the generation of mtDNA rearrangements and supports the existence of recombination hotspots.

Figure 1.

Inducible expression of Mito–ScaI–HA leads to a fast mtDNA depletion. (A) The mitochondrial localization of ScaI was confirmed by confocal microscopy of HP202B.22 cells. Colocalization of the anti-HA signal with mitotracker red was evident after induction of Mito–ScaI–HA. Cells were analyzed 24, 48 and 72 h after induction. No Mito–ScaI–HA expression was detected after 72 h induction. (B) mtDNA levels were estimated by Southern blot using two probes: one homologous to most of the mtDNA and a second one homologous to the nuclear-coded 18S rDNA. (C) The relative levels of mtDNA were expressed as a ratio to the nuclear encoded 18S rDNA. Cells were induced for 15, 30 and 60 min and collected at 0, 60 and 120 min after induction.

Figure 2.

Changes in cytochrome c oxidase activity, mtDNA/nDNA ratios and mtDNA-associated transcripts after induction of DSBs. (A) COX enzyme activity normalized against citrate synthase at 5 h, 24 h and 7 days post induction; non-induced (NI) control. (B) mtDNA/nDNA ratios determined by Southern blot, time points and controls as in panel A. (C) The mtDNA/nDNA ratios (dark gray bars) were correlated to the transcript levels of POLG, LIG3 and TFAM (filled circles in each sub-panel). All transcript levels were normalized to β-actin. The DNA and RNA samples in (C) were obtained from hepatocytes after the induction of Mito–ScaI–HA expression for 30 min and culturing for different times. The data are expressed as changes from non-induced samples (NI).

Figure 3.

mtDNA deleted molecules are generated in liver and cells after expression of Mito–ScaI–HA. (A) Agarose gel of short-extension PCR products amplified from liver of non-injected mice (NI) or at 2, 6, 10 or 14 weeks post injection of either rAd–Mito–ScaI–HA or rAd–eGFP using primers 5399F and 1078B. (B) Agarose gel of short-extension PCR products amplified from liver of non-injected mice or at 10 or 14 weeks post injection of rAd–mito–ScaI–HA using alternate primer sets indicated at the bottom of the image. (C) Agarose gel of short-extension PCR products amplified from non-induced and induced HP202B.22 cells at 2, 6 or 9 days post induction using primers 5399F and 1078B. Regions of the gels in A, B or C that were cloned and sequenced are indicated with dashed brackets.

Figure 4.

Schematic representation of the identified mtDNA deletions after multiple DSBs. Two groups of recombined molecules were identified after expression of Mito–ScaI–HA. Recombined molecules (deletions) identified with primers between 5399 and 1078 are shown in white and those identified with primers between 8300 and 15 065 are shown in gray. The preserved regions of the mtDNA are depicted as solid bars. Deletions found in liver are shown in the upper panel and those identified in cells in the lower panel. The mtDNA genetic map on top depicts the location of ScaI sites in both NZB and BALB mtDNA haplotypes, the origins of light strand (O_L) and heavy strand replication (O_H) and primer binding sites (arrows with primer names).

Figure 5.

Intramolecular and intermolecular recombination after DSBs. Twenty-seven different patterns of deleted molecules were identified in liver (panels A and B) and cells (panels C and D) after expression of Mito–ScaI–HA. Arrows indicate the extent of the deletion, nucleotide numbering is shown above each sequence. mtDNA haplotypes involved in each recombination event are indicated on the right, NZB, BALB or ND, not determined. Deletions 1–7 (A) and 8–14 (B), respectively, correspond to the white and gray bars in the upper panel if Figure 4; deletions 15–23 (C) and 24–27 (D), respectively, correspond to the white and gray bars the lower panel of Figure 4.