Twinkle has been shown to be the replicative DNA helicase in mitochondria of eukaryotic cells, and mutations that abolish expression of this gene are lethal in animal cells [6, 14, 15, 26]. Twinkle is a homologue of the bacteriophage T7 gp4 protein, which has both DNA primase and DNA helicase activities and contains the highly characterized TOPRIM domain that is conserved in DNA primases, topoisomerases and OLD family nucleases . However, until the present work no Twinkle homologue from a higher eukaryote has been shown to have DNA primase activity. Shutt and Gray have analyzed the sequence of Twinkle homologues from several eukaryote species and have proposed that in addition to being the DNA helicase, Twinkle may also serve as the mitochondrial DNA primase in most eukaryotes except metazoa . As far as we know our present report is the first to show that the Twinkle homologue in a plant species (Arabidopsis) has both DNA primase and DNA helicase activities. Other than the truncated primase homologue already mentioned (At1g30660; but there is no information available about whether this protein is functional) no other bacterial or phage-type DNA primase homologues have been found in the Arabidopsis genome sequence.
Sequence analysis provides an explanation of why the plant homologue has both activities while the animal homologues lack DNA primase activity (Figure 6). The absence of primase activity in human Twinkle is likely due to the lack of the zinc finger motifs formed by 4 cysteine residues near the N-terminal end of the protein, as well as other amino acid sequence differences at conserved sequences in the primase domain of the protein which have been shown to be responsible for the primase activity (Figure 6) . Sequence variation occurs in other metazoan species, and while some have the zinc fingers, they have differences at other conserved motifs. The Arabidopsis homologue, in contrast, retains all conserved motifs . Phylogenetic analysis further supports these findings, indicating that the plant Twinkle homologues are most closely related to the T7 gp4 protein, while the animal homologues are quite distantly related. These results suggest that the bifunctional T7 gp4 homologue may be conserved in higher plants.
The Arabidopsis Twinkle protein may function both in mitochondria and chloroplasts, as this protein has been shown to be dual-targeted to both organelles [27, 28]. These reports are based on the analysis of predicted N-terminal targeting sequences of a number of nuclear-encoded Arabidopsis proteins fused with the GFP coding region. However, it has been shown that targeting of fusion proteins can be affected by the context of the N-terminal sequence with the GFP sequence [28, 29]. A recent report on the maize plastid proteome has shown the presence of Twinkle in the chloroplast nucleoid .
Mitochondrial genomes range widely in size, from about 16.5 kbp in vertebrates and invertebrates, to 70–100 kbp in yeast and 200–2000 kbp in plants. The replication of animal mtDNA has been characterized in great detail, and in the original model each strand of the duplex DNA replicates at a different time, with the initial replication primed by a short transcript synthesized by the mitochondrial RNA polymerase . The second strand replicates only when it becomes single stranded by progression of the first strand, allowing formation of a characteristic structure to facilitate replication initiation of this strand. In yeast and plants, mtDNA replication appears to be more complex, and may involve a recombination-dependent replication mechanism [23, 31–34]. In this case DNA priming may not be required if invading strands provide the priming function for DNA synthesis. However, even in phage systems that replicate by a recombination mechanism a DNA primase is still required for priming synthesis at lagging strands during some phases of DNA replication .
A distinct mtDNA primase activity has been reported in some animal and protist cells and mtDNA primase activity has been reported in human cells, but no distinct human protein with this activity has yet been identified. It has been suggested that the DNA primase in animal cells is tightly associated with the mtDNA (γ) polymerase, and is thus difficult to isolate separately . In a trypanosome a mtDNA primase of 70 kDa has been reported , while in yeast a mtDNA primase of 67 kDa has been characterized , which are both close to the size of T7 gp4 and Twinkle. Our understanding of animal mtDNA replication is complicated by reports of strand-coupled bidirectional replication from a single replication origin, which by its nature should require a DNA primase to synthesize primers for the lagging strand [30, 38, 39]. It is unclear whether a separate mtDNA primase is present or required in species (including human) with highly compact mitochondrial genomes . Recently it has been shown that in vitro, human mitochondrial RNA polymerase is responsible for priming lagging strand mtDNA synthesis. It may be possible that priming of replication of the small animal mitochondrial genome is provided by short transcripts synthesized by the mitochondrial RNA polymerase [40, 41].
A DNA primase has been purified and characterized from pea chloroplasts , and primers synthesized by that preparation are similar in size to primers synthesized by the purified Arabidopsis Twinkle homologue. The pea enzyme is larger (~90 kDa) than the Arabidopsis Twinkle homologue, but it was not characterized for DNA helicase activity. CtDNA replication involves multiple replication origins and bidirectional DNA synthesis [42, 43], which would require DNA primase activity for lagging strand synthesis.
Organelle DNA replication appears to be different in plants (as compared to animals), which have very large and complex mitochondrial genomes and likely require multiple sites of lagging strand DNA synthesis. The role of recombination-mediated replication [33, 34] may reduce the need for primase-synthesized primers for organelle DNA replication, as an invading DNA strand could provide the 3′ ends for DNA synthesis. However, even in this case it is likely that organelle DNA primase is required in plants. Bacteriophage T4 replicates by multiple mechanisms, including recombination-dependent replication, and requires a DNA primase. The observations that the Arabidopsis Twinkle protein is expressed at highest levels in the shoot apex and other developing tissues including young leaves provides strong support for a role of the Twinkle homologue in plant organelle DNA replication, similar to its role in other species [4, 5].
Mutations in human Twinkle have been shown to lead to a drastic reduction in mtDNA copy number and disease . RNAi-mediated reduction of Twinkle expression in cultured human cells was found to lead to a rapid drop in mtDNA copy number, while overexpression of Twinkle in mouse tissue was associated with an increase in mtDNA copy number [15, 26]. In each of these cases the effect has been associated with the DNA helicase activity of the protein. We showed that this single protein from Arabidopsis has both DNA primase and DNA helicase activities in vitro, the same activity as the bacteriophage T7 gp4 protein.