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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.
6.4 A hypothesis for origin independent COSCOFA replication in
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Figure 6.4. DraI, ND5 (nt 12272-16011) and BclI, ND2 (3659-7658) digests of HEK293T mtDNA showing S1 resistant full length bubble arcs in both fragments.
It is possible that this replication mode could be initiated by transcription terminating elsewhere in the genome than in the NCR and forming an R-loop to initiate replication. Because this initiation does not result in RITOLS replication but in the more conventional strand-coupled (COSCOFA) mode, it is likely that the initiation mechanisms involved are not the same. Based on the previous examples, it is tempting to suggest that the best way to initiate DNA replication in an origin-independent manner would be RDR. Strand-invasion would be carried out by randomly-cut linear molecules resulting from double-strand breaks caused by aborted RITOLS replication or external damage. The fact that origin-independent replication dominates after mtDNA depletion by replication-inhibiting drugs, such as ddC or EtBr, supports the idea: RITOLS replication initiating from OH is disturbed and the replication forks stall before reaching the terminus. Stalled replication forks need to be resolved, resulting in double-strand breaks (Figure 6.5) or fork regression, generating dsDNA linears. In response, there might more initiation of replication at OH, but these forks would stall fairly soon resulting in the accumulation of rather short linear molecules originating from OH. Either end of these molecules could then strand-invade another mtDNA molecule and initiate replication either at OH or further away. Drug-induced replication stalling would result in randomly terminated linear molecules, the hotspots for the distal end would statistically locate close to but not at OH, but would be dispersed over a wide area and difficult to map. This would be detected as the so-called origin zone (Ori Z) of COSCOFA replication (Bowmaker et al. 2003). In reamplifying cells, the main origin zone is reported to be quite discrete and located only a short distance downstream from OH, in TAS-mt5 region ([nt 16197] Yasukawa et al. 2005). As the site was mapped using LM-PCR to detect free 5´-ends of the lagging-strand, it may well be that this “origin” is actually the dominant pause site seen in this region of the HincII digest (13637-1008, see III, Figure 4C:vi, pause site h). Degradation of broken lagging-strand linear molecules of RITOLS replication results in molecules with extensive single-stranded 3´-ends
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capable of strand-invasion and replication priming (Figure 6.5). This invading linear molecule could later be cleaved by an endonuclease as known for T4 phage, replication generating theta-like forms indistinguishable from a standard replication bubble.
Figure 6.5. A hypothetical recombination-initiated mtDNA replication mechanism in cultured cells.
A replication stall results in a double-strand break. As the lagging-strand is likely to be cleaved due to the discontinuity directly at the replication fork, resulting linear fragments could be easily degraded exposing a single-stranded 3´-end. The free 3´-end could strand invade homologous sequences in other molecules and initiate replication.
A supporting piece of evidence comes from the investigation of mtDNA from Twinkle K421A-expressing cells. This artificial mutation abolishes helicase activity and results in a strong replication-stalling phenotype with around 20% of the molecules containing stalled replication forks.
The situation is somewhat analogous to the drug-induced replication stall. 2DNAGE of mtDNA from these cells shows bubble-arcs that extend all over the HincII OH containing fragment (13637- 1008 [Figure 5.13]), and molecules where the arms of the replication fork map outside OH (Figure 5.22B) are observed by TEM, both findings indicating random replication initiation. Of nineteen replicating molecules examined from K421A Twinkle-expressing cells, one had a linear molecule protruding out from the middle of a theta replication intermediate (Figure 5.21). Closer examination showed that the linear is genuinely connected by a junction and is not just a separate linear molecule (see insert in Figure 5.21). The distance of the replication forks from the branch point is equal and the distal end of the linear arm would map outside of the bubble region, if the sequences are assumed to be homologous. If the linear fragment stems from an earlier terminated replication
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event, the two replication events should have different origins. Moreover, one fork from the second replication event would have passed the branch point without interference, which intuitively seems impossible. Although this is still an anecdotal observation, it is very difficult to postulate any other explanation for such a structure, except that of a linear molecule invading the circle and initiating bidirectional replication from the initiation point, as in T4 phage and illustrated in Figure 6.5.
Also in line with RDR as the primary mechanism of origin-independent replication, mitochondrially-targeted MnSOD-RecA seems to enhance S1 resistant replication intermediates in cultured cells, as expected for an enzyme that facilitates strand-invasion. In contrast, RecA does not alter the X-form abundance bringing further support to the idea of a noncrossover recombination mechanism in cultured cells (Figure 5.25B). Alternatively this might result from a similar DNA replication inhibition effect by a DNA binding protein as seen with TFAM overexpression. If RDR exists in all tissues, it might, most of the time, function as a backup mechanism against short mtDNA fragments that mostly arise from aborted replication replication events, whereas regular DSBs are either repaired by the synthesis-dependent strand annealing or crossover recombination. It should be noted that once the RDR has reached the end of the molecule the result is indistinguishable from synthesis-dependent strand annealing recombination. Clearly further studies are needed in order to resolve this issue.