Previous studies on organellar DNA polymerases with no gene identification in plants and algae. cp, chloroplast; mt, mitochondrion; NEM,
1. Introduction
Mitochondria and plastids are eukaryotic organelles that possess their own genomes. The existence of organellar genomes is explained by the endosymbiotic theory [1], which holds that mitochondria and plastids originated from α-proteobacteria-like and cyanobacteria-like organisms, respectively [2,3]. Organellar genomes are duplicated by the replication machinery, including DNA polymerase, of the each organelle. The enzymes involved in the replication of organellar genomes are thought to be encoded by the nuclear genome and transported to the organelles after synthesis [4].
DNA polymerase γ (Polγ) is the enzyme responsible for replicating the mitochondrial genome in fungi and animals [5,6]. Polγ belongs to family A DNA polymerases, which share sequence similarity to DNA polymerase I (PolI) of
In the late half of the 1960s, the presence of organellar DNA polymerase was confirmed by the measurement of DNA synthesis activity in isolated plant chloroplasts [7,8] and mitochondria of yeast and animals [9,10]. Since the 1970s, DNA polymerases have been purified from the chloroplasts and mitochondria of various photosynthetic organisms (Table 1), with biochemical data suggesting that plant organellar DNA polymerases and γ-type DNA polymerases share similarities with respect to optimal enzymatic conditions, resistance to aphidicolin (an inhibitor of DNA polymerase α, δ, and ε), sensitivity to NEM, molecular size, and template preference. Despite such observation, no gene encoding a homolog of Polγ has been found in the sequenced genomes of bikonts, including plants and protists. Therefore, the DNA polymerase of both mitochondria and plastids in photosynthetic organisms had remained unidentified. Sakai and colleagues [11-13] isolated nucleoid-enriched fractions from chloroplasts and mitochondria of tobacco leaves. They detected DNA synthetic activity in the nucleoid fraction and showed that the apparent molecular mass of the polypeptide exhibiting the activity was similar to Klenow fragment of DNA polymerase I (PolI) in
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1973 |
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7.2 | 6 | 10-15 | |||
1979 | Wheat (mt) b | 110m | 7 | 5 | 150 | 5 | yes |
1980 | Cauliflower (mt) c | 150 | 1 | ||||
1980 | Spinach (cp) d | 105n | 8-9 | 0.1-1 | 100 | 2 | |
1981 | Wheat (mt) e | 180m | 8 | no | |||
1984 | Pea (cp) f | 87m | 12 | 120 | 1 | no | |
1990 | Soybean (cp & mt) g | 85-90n | 8 | 125 | strongly | ||
1991 | Spinach (cp) h | 105n | 1 | yes | |||
1991 |
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110n | 100 | 2 | no | ||
1993 | Chenopodium (mt) j | 80-90n | 10 | 125 | 1 | yes | |
1995 | Soybean (cp) k | yes | |||||
2002 | Pea (cp) l | 70n | 7.5 | 8 | 125 | partially | yes |
2. Enzymatic characteristics of POPs
The isolation of POP was first reported in rice (
2.1. Properties of DNA polymerase activity
The properties of DNA polymerase activity of POPs have been examined using recombinant [27,28,31] or native proteins purified from
2.2. Processivity
Processivity is defined as the number of nucleotides added by a DNA polymerase per one binding with the template DNA. POPs, in general, have high processivity values; for example, the processivity of rice recombinant GST-POP and
2.3. Sensitivity to inhibitors
The effects of inhibitors, such as aphidicolin, NEM, dideoxyTTP (ddTTP), and phosphonoacetate (PAA), on the DNA synthesis activity of POPs were evaluated [27,31-33]. Aphidicolin is a specific inhibitor of DNA polymerases α, δ, and ε and acts through competition with dCTP or dTTP [36,37]. The sulfhydryl reagent NEM inhibits DNA polymerases α, γ, δ, and ε [38], and has a half maximal inhibitory concentration (IC50) of <0.1 mM for Polγ. PAA is an analog of pyrophosphate and interacts with viral DNA polymerases and reverse transcriptases at pyrophosphate binding sites to create an alternative reaction pathway [39,40]. ddTTP severely inhibits DNA polymerases β and γ, but only weakly impairs the activities of DNA polymerases δ and ε [41]. POPs are not inhibited by aphidicolin or NEM. The inhibitory effect of ddTTP differs depending on the organism, with the IC50 ranging from 4-615 μM for POPs (Figure 2A). The activity of POPs is severely inhibited by PAA, as demonstrated by IC50 values of 1-25 μM for several POPs (Figure 2B, C). In contrast, other family A DNA polymerases, including PolI and Polγ, are not markedly inhibited by PAA, suggesting that PAA is a useful marker for the classification of organellar DNA polymerases in unsequenced eukaryotes. T4 DNA polymerase and DNA polymerase δ of
2.4. 3'-5' Exonuclease activity
POPs have a 3'-5' exonuclease domain containing three conserved regions, Exo I, Exo II, and Exo III (Figure 1), and this exonuclease activity has been demonstrated in rice [28] and
2.5. Subcellular localization
POP was first isolated as a plastidial DNA polymerase in rice, and its localization was confirmed by immunoblot analysis using isolated plastids [27]. Subsequent studies using GFP-fusion proteins and/or immunoblotting with isolated plastids and mitochondria demonstrated that POPs are localized to both plastids and mitochondria in
2.6. The role of POP in vivo
POPs exhibit high processivity and 3'-5' exonuclease activity, and were originally thought to function as organellar DNA replicases. This speculation was verified by analyzing
3. Role of POPs in cell-cycle regulation
3.1. Organellar genome replication in plant tissues
Nuclear genomes are replicated during the DNA synthesis phase (S phase), with the daughter genomes being distributed at the mitotic phase (M phase) to maintain ploidy levels. Observations of mitochondrial DNA stained with 4',6-diamidino-2-phenylindole (DAPI) and microautoradiography using [3H]thymidine have demonstrated that the DNA content and synthesis activity in mitochondria change dramatically during cell proliferation. In the root apical meristem of geranium (
3.2. Expression of POP in plants
The spatial expression patterns of POPs were analyzed in
3.3. Red algal cell cycle
The unicellular red alga
We have also determined the replication phases of nuclear, plastid, and mitochondrial DNA by quantitative PCR using cyanobacterial DNA as an internal standard to estimate the absolute amount of DNA (Figure 4, [57]). In the first cell cycle pattern, the level of nuclear and organellar DNA was unaltered (Figure 4A). Nuclear DNA replicated at or near the M-phase in the second and third cycles (Figure 4B, C). The replication of the mitochondrial genome was synchronized with the cell cycle to some extent, with mitochondrial DNA beginning to increase from the middle (second cycle) or beginning (third cycle) of the light phase, and doubling at or near the M-phase, as was observed for nuclear DNA (Figure 4B, C). In contrast, plastid DNA replication continued throughout the entire cell cycle, even after cell division was complete (Figure 4B, C). These results suggest that the replication of nuclear and organellar DNA is initiated after the accumulation of sufficient nutrients by photosynthesis, and that light alone does not serve as a replication signal for nuclear or organellar genomes. Therefore,
3.4. Expression of POP in the red algal cell cycle
We determined the expression of POP in synchronous culture of
The transcript level of other possible genes related to organellar DNA replication in
4. Possible evolutionary history of organellar DNA polymerases in eukaryotes
POP belongs to family A DNA polymerases, consisting of polymerases harboring sequence similarity to bacterial PolI, such as Polγ, DNA polymerase θ (Polθ), DNA polymerase ν (Polν), and PREX (plastid replication and repair enzyme complex, [64]). Polθ and Polν are DNA repair enzymes and are localized to the nucleus [65,66]. PREX is an apicoplast (plastid like organelle)-localized DNA polymerase in the malaria parasite
Figure 5 shows a phylogenetic tree of family A DNA polymerases. From the tree, it is clear that POPs belong to a well-defined clade that is evolutionarily separated from bacterial PolI. Therefore, it can be concluded that POPs did not originate from PolI of cyanobacteria nor α-proteobacteria. Although PREX may have originated from a red algal secondary endosymbiont, their origin remains unclear, because PREX do not contain POP-specific sequences (Figure 8). POPs are widely conserved in eukaryotes, including amoebozoa, that have a close relationship with opisthokonts in phylogenetic analyses, but POPs have not been detected in opisthokonts, including animals and fungi (Figure 6). This suggests that POP might have originated before the diversification of photosynthetic eukaryotes. Pathogenic protists of animals, including
From the phylogenetic tree, we proposed an evolutionary model of organellar DNA polymerases (Figure 7). Initially, when the ancestor of eukaryotes acquired mitochondria, the elementary mitochondrial replicase was likely bacterial DNA polymerase III (PolIII) (1 in Figure 7A). PolIII was then replaced by a POP, and the host cell then used POP for the replication of organellar genomes (2 in Figure 7A). We presume that PolIII must have been introduced upon the endosymbiosis event, but another possibility is that an endosymbiont or a host cell had already possessed POP before endosymbiosis. But this idea is considered unlikely because no bacteria having POP have been found so far. In this respect, it is of interest to note that, based on phylogenetic analysis in family A DNA polymerases, it has been postulated that Polγ is of phage origin [69]. POP could also have been acquired from a virus. In effect, the ultimate origin of the ancestral POP is still unknown. The phylogenetic tree (Fig. 5) suggests that the closest relative of POP is Polν or Polθ, which are present in various eukaryotes. It is not impossible then that an ancestral polymerase in eukaryotic host diverged into POP, Polν and Polθ.
In the plastids of plants and algae, POP also replaced PolIII, and thus POPs are presently found in most eukaryotes (3-5 and 6-8 in Figure 7A). In opisthokonts, however, POP was replaced by Polγ, whose origin is also unknown (4 in Figure 7A). Chromalveolates, consisting of alveolates and heterokonts such as diatoms, must have had a POP for mitochondrial replication before the occurrence of secondary endosymbiosis. Phylogenetic analysis suggests that the POPs of diatoms are more closely related to red algal POP than the POPs of ciliate
Based on the genomic data obtained to date, Polγ is found only in opisthokonts, indicating that two different polymerases cannot co-exist, at least over a long evolutionary span. The catalytic subunit of bacterial PolIII is also not encoded by eukaryotic genomes, although the PolIII gamma subunit, which functions as a clamp loader in bacteria, is conserved in land plants, such as
5. Conclusion and prospects
POPs have been isolated as organellar-specific DNA polymerases in a number of photosynthetic eukaryotes and ciliates. As the majority of biologists still believe that all mitochondrial replication enzymes are Polγ, the primary objective of this review was to introduce POP to the wider research community. Although both POP and Polγ are family A DNA polymerases, their primary structures are quite different from one another. However, POP and Polγ display similar DNA polymerase activities that are characteristics of replicases, including high processivity, 3′-5′ exonuclease activity, and reverse transcriptase activity. Eukaryotes containing a POP gene do not have a gene for Polγ, and
The sensitivity of POP to DNA polymerase inhibitors clearly differs from that of Polγ. To date, POPs have been shown to be commonly inhibited by phosphonoacetate. The inhibition mechanisms remained unclear for family A DNA polymerases, including POP, although it was reported that motif A in the polymerase domain of family B DNA polymerases is involved in the sensitivity to phosphonoacetate [42,43]. The detailed study of the inhibitory mechanisms and structural analysis of POP are needed, although POP is likely to be conserved in pathogenic bikonts, such as the green alga
In multicellular plants, genomes of organelles are replicated in meristematic tissues, but the process is not synchronous with the cell cycle or even with organellar division. In the unicellular red alga
Acknowledgments
This work was supported in part by Grants from Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST), Japan, the Global Center of Excellence (GCOE) Program “From the Earth to ‘Earths’” from the MEXT, Japan, and the Canon Foundation.Appendix
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