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. 2005 Apr 1;14(7):893-902.
doi: 10.1093/hmg/ddi082. Epub 2005 Feb 9.

Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans

Sarika Srivastava  1 , Carlos T Moraes
Affiliations

Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans

Sarika Srivastava et al. Hum Mol Genet. .

Abstract

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders and have been found to accumulate during normal aging. Despite the fact that hundreds of deletions have been characterized at the molecular level, their mechanisms of genesis are unknown. We tested the effect of double-strand breaks of muscle mtDNA by developing a mouse model in which a mitochondrially targeted restriction endonuclease (PstI) was expressed in skeletal muscle of mice. Because mouse mtDNA harbors two PstI sites, transgenic founders developed a mitochondrial myopathy associated with mtDNA depletion. The founders showed a chimeric pattern of transgene expression and their residual level of wild-type mtDNA in muscle was approximately 40% of controls. We were able to identify the formation of large mtDNA deletions in muscle of transgenic mice. A family of mtDNA deletions was identified, and most of these rearrangements involved one of the PstI sites and the 3' end of the D-loop region. The deletions had no or small direct repeats at the breakpoint region. These features are essentially identical to the ones observed in humans with multiple mtDNA deletions in muscle, suggesting that double-strand DNA breaks mediate the formation of large mtDNA deletions.

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Figures

Figure 1
Figure 1. Production of transgenic mice expressing a mitochondrial-targeted PstI in skeletal muscle.
Panel a depicts the structure of the transgene construct containing a mammalianized version of the bacterial PstI gene fused in frame with an N-terminal mitochondrial targeting sequence of COX VIII. Panel b shows founder 1 (Fd1) and a non-transgenic sibling. Extensive breeding of Fd1 and Fd2 failed to produce pups containing the transgene, whereas 50% of Fd3 pups had the transgene (Panel c). Western blot analysis showed that mitochondria isolated from Fd1 and Fd2 skeletal muscle (fore limb and hind limb muscles) contained the transgene product. Muscle mitochondria from a Fd3 pup harboring the transgene did not express PstI (panel d). Muscle mitochondria isolated from Fd1, Fd2 and non-transgenic littermate controls were analyzed for the activity of complex I+III, II+III and IV by spectrophotometric assays (panel e). The activities were expressed as a ratio to citrate synthase activity to normalize the results to mitochondrial abundance. Complexes I and IV were significantly decreased in the transgenic Fd1 and Fd2. *p<0.05; **p<0.005; n=3 independent assays of each mitochondrial preparation.
Figure 2
Figure 2. Chimeric expression of the Mito-PstI in muscle of Fd1 and Fd2.
Panel a shows histochemical staining for cytochrome oxidase (COX) and succinate dehydrogenase (SDH) activities. Both Fd1 and Fd2 had COX deficiency but normal or elevated SDH activity. The COX deficiency was present in a mosaic pattern. The mosaic pattern of expression of Mito-PstI was also observed by immunocytochemistry using an anti-PstI antibody (Panel b). Fibers expressing Mito-PstI had decreased levels of CoxIp, a mtDNA-coded polypeptide (panel b).
Figure S2
Figure S2
Transmission electron microscopy showed mitochondrial proliferation and swelling in muscle fibers of Fd1 (right panel). Mitochondrial cristae disorganization can also be observed in Fd1 muscle.
Figure 3
Figure 3. Detection of mtDNA depletion and mtDNA deletions in muscle of Mito-PstI transgenic animals.
Southern blot analyses of DNA extracted from skeletal muscle of Fd1 and Fd2 was performed with mtDNA probes [COXI (panels c, d and f) and ND4 regions (panel e)] and the nuclear 18S rDNA (panel b). A similar analysis was also performed for DNA extracted from liver of transgenic founders (panels g-i). Total muscle DNA was either digested with NheI (most panels), or analyzed undigested (panel f). These studies showed a muscle specific mtDNA depletion (an approximate 60–70% reduction) in the founders (panel j). The Southern analysis also showed the presence of molecules migrating faster than the wild-type mtDNA in muscle of Fd1 and Fd2 (panel a, COXI probe), which could correspond to partially-deleted mtDNAs (ΔmtDNA). The undigested sample (panel f), showed the presence of bands that were tentatively assigned as, wild-type circles (cWT); linearized “broken” circles (lWT); and circular partially-deleted molecules (cΔ). A PstI-digested fragment would migrate at 12.5 kbp, and could be present at low levels.
Figure 4
Figure 4. Characterization of mtDNA deletions associated with a double-strand break.
Using oligonucleotide primers flanking a PstI site and the D-loop region (arrows in panel a), we amplified an approximate 650 bp PCR fragment only in DNA extracted from muscle of Fd1 and Fd2 (panel b). The PCR products were cloned into plasmids and sequenced. Sequence analysis showed the presence of a family of mtDNA deletions involving the PstI site region and the end of the D-loop (panel c). Most deletions had small homologies at the breakpoint region (underlined nucleotides in panel c).
Figure 5
Figure 5. Clonal expansion of mtDNA deletions in muscle.
Muscle sections of transgenic founders were stained for cytochrome oxidase activity and dissected by Laser Capture Microscopy. Panel A shows a nested PCR for the mtDNA deleted region breakpoint of representative COX-negative fiber segments. Positive reactions were cloned into plasmids and digested with EcoRI to release the insert (panel B). Sequencing reactions identified the deletion breakpoints, which were coded as in figure 4.
Figure 6
Figure 6. A model for the formation of Class II deletions.
Because of the molecular similarities between the PstI generated deletions and class II deletions in humans, we propose that double-strand breaks could be a generator of these type of rearrangements. There are at least two potential mechanisms: The free 3′ end produced by PstI could anneal at the end of the D-loop region with a the participation of small repeats (Single-Strand Annealing, SSA) and function as a replication primer, extending the strand back into the D-loop region and beyond (panel a). Alternatively, polymerase pauses during replication would expose single-strand regions that could be annealed to the free ′ end of the cut DNA (panel b).
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