Replication of animal (vertebrate) mtDNA

The exact mechanism of mitochondrial DNA replication in animals has caused controversy in recent years. The mtDNA replication mechanism from insects to mammals has been thought to be very similar. However most recent work has concentrated only on mammalian and avian tissues and cultured cells. An intense debate of the exact replication mechanism(s) is still ongoing. Some of the work of this thesis was done to address the issue, including the original articles II and submitted manuscript IV. The submitted manuscript IV will also bring forth evidence of a novel replication-

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related mechanism operating in some human cells. The proposed mechanism and their consequences thus will be discussed in more detail in the discussion section of this thesis.

2.14.6.1 Strand-displacement model (SDM)

The long prevailing paradigm in the field describes an unusual mode of strand-displacement replication, where leading- and lagging-strands are synthesized asymmetrically and unidirectionally from separate and well-defined origins, OH (heavy- or leading-strand origin) and OL (light- or lagging-strand origin) (Robberson et al. 1972, Robberson & Clayton 1972, Crews et al. 1979, Clayton 1982).

Replication starts from the OH origin and is primed by a processed transcript from the light-strand promoter (LSP). This processed RNA strand is highly persistent, forming an R-loop structure in the non-coding region of mtDNA (Shadel & Clayton 1997, Lee & Clayton 1998). The model elegantly links mitochondrial transcription to replication and provides a possible mechanistic way for mtDNA proliferation to respond to various stimuli.

The endonucleolytic processing of the LSP transcript is proposed to be carried out by a site-specific endonuclease, RNase MRP, which is capable of cleaving transcripts sequences spanning from CSB II to CSB III. Interestingly, the substrate specificity of RNase MRP resides in an RNA component encoded by a nuclear gene. Similar ribonucleoproteins are known also from yeast and the frog Xenopus laevis. These conclusions have been questioned and instead it has been proposed that CSBII functions as a powerful transcription terminator sequence (Falkenberg et al.

2007). Thus the H-strand primer formation could be explained by a protein-modulated transcription termination at CSB II.

The DNA polymerase PolG replicates the nascent H-strand, and with the help of a helicase, displaces the parental H-strand while processing. Consequently, the single-stranded displaced strand is protected by single-strand binding proteins (SSB) from degradation (Figure 2.15). When the replication reaches two-thirds of the genome, OL initiates. OL is flanked by tRNA clusters and it is suggested that when released in single-stranded form, it tends to form a hairpin structure. A putative primase is proposed to recognise the hairpin and generate an RNA primer for the lagging-strand, after which L-strand replication begins. However, the evidence for such a primase is weak. After replication, the primers are removed by RNase H activity and gaps filled.

More recently, additional L-strand origins have been suggested (Brown et al. 2005). It is not known

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how these origins are primed, but they could enable faster dsDNA transition of lagging-strand replication intermediates.

Figure 2.15. The strand displacement model of mammalian mtDNA replication. The leading-strand replication initiates from OH and proceeds two-thirds of the genome before the lagging-strand replication is initiated from OL.

2.14.6.2 Strand-synchronous model

Studies involving two-dimensional neutral-neutral agarose gel electrophoresis (2DNAGE) methodology have led to the proposal of a strand-coupled mechanism operating from less discrete origins (Holt et al. 2000, Yang et al. 2002, Bowmaker et al. 2003, Yasukawa et al. 2005, Yasukawa et al. 2006). This mechanism basically involves theta-replication and was originally suggested to function as an alternative to the strand displacement model. Initiation and priming of this replication mode remain unclear. However, there are no mechanistic constraints why the initiation and priming for the leading-strand as described for the SDM would not apply.

One peculiarity of the model is that the lagging-strand DNA is synthesized with delay and the newly replicated lagging-strand is originally laid down as RNA. As a result the RNA rich mode of replication can be detected as RITOLS (Ribonucleotide Incorporation ThroughOut the

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Lagging-Strand) intermediates (Figure 2.16). RITOLS intermediates are subsequently replaced by or matured to DNA. In this model OH remains the prominent replication origin for the leading- strand, but also functions as the terminus. Replication originating from O_H proceeds unidirectionally through the genome. RITOLS replication intermediates (RIs) are prone to RNase H degradation during mtDNA extraction, resulting in single-stranded parental H-strand, explaining the observations that lead to the generation of the strand-asynchronous model (Yang et al. 2002).

Figure 2.16. RITOLS replication of mammalian mtDNA. Strand-coupled, unidirectional replication initiates from OH. Initially the lagging-strand is laid down as RNA, but is subsequently matured to DNA.

Considering lagging-strand synthesis, the mechanism results in superficially similar molecules as the displacement model, although unlike in the SDM, the lagging-strand also terminates at OH. The key challenge in the future will be to show what mechanisms are responsible for persistent RNA formation on the lagging-strand and how the lagging-strand maturation or synthesis is initiated. Based on the same methodology as the previous observations, it seems that a minority of mtDNA molecules take advantage of a more COnventional, Strand-Coupled Okazaki Fragment-Associated replication (COSCOFA) that has no persistent RNA-DNA hybrid formation and is initiated from a broader zone (Holt et al. 2000, Yasukawa 2005). This replication mode seems to be preferred in cultured cells that are amplifying their mtDNA after drug-induced depletion. The replication initiating from this zone starts bidirectionally, but because initiation occurs close to the OH the replication still proceeds through most of the molecule in an apparently unidirectional way (Figure 2.17).

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Figure 2.17. COSCOFA replication of mtDNA. Bidirectional and strand-coupled replication initiates from a broader zone (ori-z). Replication proceeding to the OH direction terminates at OH, resulting in unidirectional replication of the rest of the molecule. The lagging-strand synthesis does not involve extensive RNA runs.

In rodent and chick liver as well as in untreated cultured human cells RITOLS intermediates are the predominant class (Holt et al. 2000, Yasukawa et al. 2005, Yasukawa et al.

2006).