In contrast to most tissues and cultured cells, human cardiac muscle mtDNA has abundant recombination intermediates as well as conventional Y-arc dsDNA replication intermediates.
Furthermore, half of the mtDNA molecules seem to be dimeric, which is quite an unusual feature for healthy tissues. When analysed in detail with 2DNAGE and TEM, no standard theta-like replication intermediates can be found. Recombination intermediates are also fairly abundant in human brain, but they coexist with theta-like intermediates and there are many fewer dimeric molecules. As brain is a heterogeneous tissue of several cell types, it is likely that these molecules represent mtRIs from different cells rather than several processes happening in the same
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mitochondria. Low amounts of recombination intermediates can also be detected in other mammalian tissues and cultured cells (Figure 5.7, Figure 2 in IV).
Interestingly, the high levels of dimers and recombination intermediates seem to be quite specific for human heart, they cannot be detected in mouse, rabbit or pig heart. The only mouse tissue analysed with abundant recombination intermediates is brain. This fits together with observations of Pikó and Matsumoto (1977) that mouse brain has the highest frequency of circular dimers and complex molecular forms compared to heart, kidney and liver. Recombination of two monomeric circles would be the simplest mechanism of dimer formation.
The reason for such an active recombination in heart mtDNA is hard to interpret in the light of any conventional models of DNA maintenance. Due to homoplasmy, there is no need for genetic recombination in mtDNA. If it is a simple matter of of double-strand break repair this would imply that heart mtDNA suffers huge amounts of damage, as inducing similar levels of recombination intermediates involves heavy use of genotoxic drugs or radiation in other systems (eg. Liberi et al. 2005, Donaldson et al. 2006). Moreover, I was unable to induce such forms in cultured cells by inducing DNA damage using KBrO3. However, there is one abundant and physiologically-significant source of DNA damage in heart: reactive oxygen species (ROS) originating from the highly-active electron transport chain. Although oxidized nucleotides can be repaired effectively by other means, there is evidence that they are a major source of double-strand breaks (Lieber et al. 2003, David et al. 2007). The exact mechanism of DSB induction is unknown.
A chain reaction–like oxidation of DNA as well as interference with replication has been suggested.
The absence of standard theta replication intermediates in human cardiac muscle mtDNA, combined with the evidence that mtDNA in this tissue is organized in multimeric networks joined by abundant recombination junctions and by catentation, suggests that mammalian mtDNA might in some cases utilize recombination-dependent initiation for replication. RDR is widely established as the main mode of mtDNA replication in many other organisms as discussed earlier in chapter 2.14.3. This conclusion is supported by the observation that the replication intermediates of PvuII- or BamHI-cut human heart migrate on 2DNAGE more in the position of a double-Y arc instead of bubble- or eyebrow-arcs (Figure 5.7). When PvuII-cut mtDNA from cultured cells is treated with S1 nuclease, the bubble arc is broken and broken bubble intermediates migrate on an arc close to the standard Y-arc form (Supplementary Figure 2e in Manuscript IV). S1 treatment of a similar heart mtDNA digest reveals that no broken bubbles are generated; instead the double-Y becomes more clearly defined. Moreover, the TEM examination of heart mtDNA reveals complex molecular networks, which include forked replication intermediates (Figures 5.5, 5.6). A partial BamHI digest reveals examples of trident structures, where three arms of a four way molecule have
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equal lengths (Figure 5K in Article IV). The lengths of these arms would in most cases map one fork to the vicinity of OH. This type of structure could represent invading linear molecules that have initiated replication from the invasion point, as in T4 phage DNA replication (Figure 6.2). If the invading strand is not cut, the resulting molecules have four arms and would migrate on the double- Y arc on 2DNAGE.
Figure 6.2. Generation of a trident structure at the OH region of mtDNA based on the T4 phage replication model. Applied from Kreuzer (2000).
Based on the existing data it is impossible to say whether RDR results in the initiation of theta-like replication as in T4 (Mosig 1998, Kreuzer 2000) or rolling circle replication as in yeast (Ling & Shibata 2004). It also cannot be excluded that heart mtDNA replicates as a linear molecule.
Although longer linear molecules of more than 2n in human heart mtDNA were not detected, linear monomers and dimers would be sufficient to sustain such a mode of replication whilst avoiding the
“end attrition” problem mentioned in section 2.13.
Curiously OH seems to be the definitive replication terminus also in human heart mtDNA; few, if any, replication forks progress past it (Figure 6.3.). This observation has further consequences for the replication of dimeric molecules with two OH regions. Dimers need to initiate twice in order to become fully replicated. Optionally, rolling circle replication initiating from OH
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could replicate half of the dimer and the resulting linear could be integrated into the network, initiate another round of replication or be circularized by snap-back recombination producing monomers. In order to replicate the whole genome-sized molecule RDR must also initiate site- specifically and unidirectionally from OH. Thus the NCR of human mitochondria could play the same role as the T4 phage terminal repeats. Alternatively, dimers may not be replicated as such at all; but this would require specific molecular mechanism to distinguish DNA sequences associated with dimers from monomers, which is unlikely. It also cannot be excluded that the molecules containing replication intermediates that do not terminate at OH, contain also an X-junction, making them migrate as a more complex species on 2DNAGE.
Figure 6.3. Persistent replication terminus at OH. (A) Diagrammatic view of mtDNA showing HincII sites flanking the NCR and resulting restriction fragment with paused fork. (B) 2DNAGE pattern of a heart HincII digest, probed for the NCR fragment. (C) Interpretation of the result. Note that the Y-arc does not reach the linear arc, indicating that the replication is unidirectional and OH is the terminus also in heart mtDNA. It is possible that any readthrough molecules also contain an X- junction and could migrate somewhere else.
It should be noted that, whilst X-forms seen in heart mtDNA are a consequence of highly-active recombination, they do not necessarily represent replication intermediates.
X-junctions seem to be fairly evenly distributed around the genome, although the appearance of the X-arc in single cut 2DNAGE might indicate that there are certain hotspot regions for cross-overs.
The distinction between X-forms and replication intermediates is important in the context of TFAM and Twinkle overexpressing mice. In these mice there is a tissue-specific increase in X-forms in heart, brain and skeletal muscle. It is possible that both Twinkle and TFAM function in recombination in mammalian mitochondria. However, they do not influence the abundance of
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X-forms in cultured cells, suggesting that underlying tissue-specific molecular mechanisms are involved. The overexpression of either one of these transgenes also results in mtDNA copy number increase in mice, but not in cultured cells. In human heart the low abundance of Twinkle mRNA compared to e.g. skeletal muscle (Spelbrink et al. 2001) makes it an unlikely candidate for modulation of recombination without the involvement of other proteins. However, the possibility of tissue specific, function-affecting post-translational modifications cannot be excluded.
A possible explanation for this observation without invoking completely new activities for these proteins is that a proportion of the mtDNA in the affected tissues already replicates via RDR, but the overall copy number inside nucleoids is relatively low compared to the human heart (Dr. T. Ide, personal communication). It should be noted that wild type mice have few if any high molecular weight mtDNA forms in their mitochondria (Figure 5.27). If these forms are a representation of a more complex, higher copy number nucloid structure, this might be further evidence for a lower mtDNA copy number per nucleoid in mouse tissues compared to human.
Twinkle and TFAM could increase the copy number (maybe in independent ways), resulting in more mtDNA copies per nucleoid and providing more opportunities for strand exchange by an already existing active recombination mechanism. However, TFAM overexpression results in a much stronger effect on the intensities of the X-spike and on the loss of RITOLS intermediates than Twinkle, despite the fact that the increase in mtDNA copy number is comparable. It could be that TFAM is also involved in promoting the formation of recombination junctions, as in the case of Abf2p in yeast (Zelenaya-Troitskaya et al. 1998).
The idea of density-induced recombination is further supported by the appearance of high molecular weight mtDNA forms in affected tissues of the overexpressing mice (Figure 5.27).
A similar DNA density-related phenomenon is represented in T4, where complex molecular networks are formed only when there are many genomes present (Mosig et al. 1995, Mosig 1998, Kreuzer 2000). In the early phase of the T4 life-cycle, DNA replication is dependent on transcription, but when late-phase proteins such as endonuclease VII (endo VII) are expressed, there is a developmental switch to RDR. It has been suggested that mtDNA replication is also generally dependent on transcriptional activity (e.g. Bonawitz et al. 2006), which fits to the RITOLS model.
Expression of a T4 endo VII–like gene in some human tissues could, in theory, be sufficient to trigger a copy number boost via RDR activation. Such a process would be much more efficient than could be achieved by gradual increase in transcription without possible disadvantage of interfering with the transcription regulation. This hypothesis is substantiated by the fact that in the higher plant Chenopodium album mitochondria the formation of complex networks also depends on the cell cycle and is linked to an overall increase in mtDNA synthesis (Backert & Börner 2000). DSB
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generation could be also an alternative explanation for the TFAM and Twinkle overexpressor mice mtDNA recombination phenotype. At least in TFAM overexpressing cells there is a marked increase in the Y-forms in 2DNAGE of PvuII digested mtDNA (Figure 2b:iv-vi in I). These intermediates could represent rolling circle–like replication intermediates resulting from strand breakage at OH. It may well be that mitotic cells recircularize these intermediates via synthesis- dependent strand annealing (Figure 2.19) or by some other means, whereas post-mitotic tissues either initiate RDR or Holliday junction formation. This hypothesis should be easily testable.