Organellar DNA Polymerases Research Articles

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain

BackgroundThe mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD).ResultsHerein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase.ConclusionsWe conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

PrimPol is a novel Primase–Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3′-GTCG-5′ DNA sequence, where the 3′-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.

Plant Organellar DNA Polymerases Evolved Multifunctionality through the Acquisition of Novel Amino Acid Insertions.

The majority of DNA polymerases (DNAPs) are specialized enzymes with specific roles in DNA replication, translesion DNA synthesis (TLS), or DNA repair. The enzymatic characteristics to perform accurate DNA replication are in apparent contradiction with TLS or DNA repair abilities. For instance, replicative DNAPs incorporate nucleotides with high fidelity and processivity, whereas TLS DNAPs are low-fidelity polymerases with distributive nucleotide incorporation. Plant organelles (mitochondria and chloroplast) are replicated by family-A DNA polymerases that are both replicative and TLS DNAPs. Furthermore, plant organellar DNA polymerases from the plant model Arabidopsis thaliana (AtPOLIs) execute repair of double-stranded breaks by microhomology-mediated end-joining and perform Base Excision Repair (BER) using lyase and strand-displacement activities. AtPOLIs harbor three unique insertions in their polymerization domain that are associated with TLS, microhomology-mediated end-joining (MMEJ), strand-displacement, and lyase activities. We postulate that AtPOLIs are able to execute those different functions through the acquisition of these novel amino acid insertions, making them multifunctional enzymes able to participate in DNA replication and DNA repair.

Inventory and Evolution of Mitochondrion-localized Family A DNA Polymerases in Euglenozoa.

The order Trypanosomatida has been well studied due to its pathogenicity and the unique biology of the mitochondrion. In Trypanosoma brucei, four DNA polymerases, namely PolIA, PolIB, PolIC, and PolID, related to bacterial DNA polymerase I (PolI), were shown to be localized in mitochondria experimentally. These mitochondrion-localized DNA polymerases are phylogenetically distinct from other family A DNA polymerases, such as bacterial PolI, DNA polymerase gamma (Polγ) in human and yeasts, “plant and protist organellar DNA polymerase (POP)” in diverse eukaryotes. However, the diversity of mitochondrion-localized DNA polymerases in Euglenozoa other than Trypanosomatida is poorly understood. In this study, we discovered putative mitochondrion-localized DNA polymerases in broad members of three major classes of Euglenozoa—Kinetoplastea, Diplonemea, and Euglenida—to explore the origin and evolution of trypanosomatid PolIA-D. We unveiled distinct inventories of mitochondrion-localized DNA polymerases in the three classes: (1) PolIA is ubiquitous across the three euglenozoan classes, (2) PolIB, C, and D are restricted in kinetoplastids, (3) new types of mitochondrion-localized DNA polymerases were identified in a prokinetoplastid and diplonemids, and (4) evolutionarily distinct types of POP were found in euglenids. We finally propose scenarios to explain the inventories of mitochondrion-localized DNA polymerases in Kinetoplastea, Diplonemea, and Euglenida.

Plant organellar DNA polymerases bypass thymine glycol using two conserved lysine residues.

Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.

Plant Organelle Genome Replication.

Mitochondria and chloroplasts perform essential functions in respiration, ATP production, and photosynthesis, and both organelles contain genomes that encode only some of the proteins that are required for these functions. The proteins and mechanisms for organelle DNA replication are very similar to bacterial or phage systems. The minimal replisome may consist of DNA polymerase, a primase/helicase, and a single-stranded DNA binding protein (SSB), similar to that found in bacteriophage T7. In Arabidopsis, there are two genes for organellar DNA polymerases and multiple potential genes for SSB, but there is only one known primase/helicase protein to date. Genome copy number varies widely between type and age of plant tissues. Replication mechanisms are only poorly understood at present, and may involve multiple processes, including recombination-dependent replication (RDR) in plant mitochondria and perhaps also in chloroplasts. There are still important questions remaining as to how the genomes are maintained in new organelles, and how genome copy number is determined. This review summarizes our current understanding of these processes.

Organellar DNA Polymerases in Complex Plastid-Bearing Algae.

DNA replication in plastids and mitochondria is generally regulated by nucleus-encoded proteins. In plants and red algae, a nucleus-encoded enzyme called POP (plant and protist organellar DNA polymerase) is involved in DNA replication in both organelles by virtue of its dual localization. POPs are family A DNA polymerases, which include bacterial DNA polymerase I (PolI). POP homologs have been found in a wide range of eukaryotes, including plants, algae, and non-photosynthetic protists. However, the phylogeny and subcellular localizations of POPs remain unclear in many algae, especially in secondary and tertiary plastid-bearing groups. In this study, we report that chlorarachniophytes possess two evolutionarily distinct POPs, and fluorescent protein-tagging experiments demonstrate that they are targeted to the secondary plastids and mitochondria, respectively. The timing of DNA replication is different between the two organelles in the chlorarachniophyte Bigelowiella natans, and this seems to be correlated to the transcription of respective POP genes. Dinoflagellates also carry two distinct POP genes, possibly for their plastids and mitochondria, whereas haptophytes and ochrophytes have only one. Therefore, unlike plants, some algal groups are likely to have evolved multiple DNA polymerases for various organelles. This study provides a new insight into the evolution of organellar DNA replication in complex plastid-bearing organisms.

Plant organellar DNA polymerases repair double-stranded breaks by microhomology-mediated end-joining.

Double-stranded breaks (DSBs) in plant organelles are repaired via genomic rearrangements characterized by microhomologous repeats. These microhomologous signatures predict the existence of an unidentified enzymatic machinery capable of repairing of DSBs via microhomology-mediated end-joining (MMEJ) in plant organelles. Here, we show that organellar DNA polymerases from Arabidopsis thaliana (AtPolIA and AtPolIB) perform MMEJ using microhomologous sequences as short as six nucleotides. AtPolIs execute MMEJ by virtue of two specialized amino acid insertions located in their thumb subdomains. Single-stranded binding proteins (SSBs) unique to plants, AtWhirly2 and organellar single-stranded binding proteins (AtOSBs), hinder MMEJ, whereas canonical mitochondrial SSBs (AtmtSSB1 and AtmtSSB2) do not interfere with MMEJ. Our data predict that organellar DNA rearrangements by MMEJ are a consequence of a competition for the 3′-OH of a DSBs. If AtWhirlies or AtOSBs gain access to the single-stranded DNA (ssDNA) region of a DSB, the reaction will shift towards high-fidelity routes like homologous recombination. Conversely MMEJ would be favored if AtPolIs or AtmtSSBs interact with the DSB. AtPolIs are not phylogenetically related to metazoan mitochondrial DNA polymerases, and the ability of AtPolIs to execute MMEJ may explain the abundance of DNA rearrangements in plant organelles in comparison to animal mitochondria.

Plant organellar DNA polymerases paralogs exhibit dissimilar nucleotide incorporation fidelity.

The coding sequences of plant mitochondrial and chloroplast genomes present a lower mutation rate than the coding sequences of animal mitochondria. However, plant mitochondrial genomes frequently rearrange and present high mutation rates in their noncoding sequences. DNA replication in plant organelles is carried out by two DNA polymerases (DNAP) paralogs. In Arabidopsis thaliana at least one DNAP paralog (AtPolIA or AtPolIB) is necessary for plant viability, suggesting that both genes are partially redundant. To understand how AtPolIs replicate genomes that present low and high mutation rates, we measured their nucleotide incorporation for all 16-base pair combinations in vitro. AtPolIA presents an error rate of 7.26 × 10-5 , whereas AtPolIB has an error rate of 5.45 × 10-4 . Thus, AtPolIA and AtPolIB are 3.5 and 26-times less accurate than human mitochondrial DNAP γ. The 8-fold difference in fidelity between both AtPolIs results from a higher catalytic efficiency in AtPolIA. Both AtPolIs extend from mismatches and the fidelity of AtPolIs ranks between high fidelity and lesion bypass DNAPs. The different nucleotide incorporation fidelity between AtPolIs predicts a prevalent role of AtPolIA in DNA replication and AtPolIB in DNA repair. We hypothesize that in plant organelles, DNA mismatches generated during DNA replication are repaired via recombination-mediated or DNA mismatch repair mechanisms that selectively target the coding region and that the mismatches generated by AtPolIs may result in the frequent expansion and rearrangements present in plant mitochondrial genomes.

Identification of a unique insertion in plant organellar DNA polymerases responsible for 5′-dRP lyase and strand-displacement activities: Implications for Base Excision Repair

Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5′-deoxyribose phosphate (5′-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5′-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5′-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5′-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs.

Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence.

DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5′-(G/C)GGA-3′ template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5′-GA-3′) as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ.

Plant organellar DNA polymerases are replicative and translesion DNA synthesis polymerases.

Genomes acquire lesions that can block the replication fork and some lesions must be bypassed to allow survival. The nuclear genome of flowering plants encodes two family-A DNA polymerases (DNAPs), the result of a duplication event, that are the sole DNAPs in plant organelles. These DNAPs, dubbed Plant Organellar Polymerases (POPs), resemble the Klenow fragment of bacterial DNAP I and are not related to metazoan and fungal mitochondrial DNAPs. Herein we report that replicative POPs from the plant model Arabidopsis thaliana (AtPolI) efficiently bypass one the most insidious DNA lesions, an apurinic/apyrimidinic (AP) site. AtPolIs accomplish lesion bypass with high catalytic efficiency during nucleotide insertion and extension. Lesion bypass depends on two unique polymerization domain insertions evolutionarily unrelated to the insertions responsible for lesion bypass by DNAP θ, an analogous lesion bypass polymerase. AtPolIs exhibit an insertion fidelity that ranks between the fidelity of replicative and lesion bypass DNAPs, moderate 3′-5′ exonuclease activity and strong strand-displacement. AtPolIs are the first known example of a family-A DNAP evolved to function in both DNA replication and lesion bypass. The lesion bypass capabilities of POPs may be required to prevent replication fork collapse in plant organelles.

Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants.

Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles.

Plastid nucleoids: evolutionary reconstruction of a DNA/protein structure with prokaryotic ancestry.

Plastid nucleoids: evolutionary reconstruction of a DNA/protein structure with prokaryotic ancestry.

Localization and Phylogenetic Analysis of Enzymes Related to Organellar Genome Replication in the Unicellular Rhodophyte Cyanidioschyzon merolae

Plants and algae possess plastids and mitochondria harboring their own genomes, which are replicated by the apparatus consisting of DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, and primer removal enzyme. In the higher plant Arabidopsis thaliana, organellar replication-related enzymes (OREs) are similar in plastids and mitochondria because many of them are dually targeted to plastids and mitochondria. In the red algae, there is a report about a DNA replicase, plant/protist organellar DNA polymerase, which is localized to both plastids and mitochondria. However, other OREs remain unclear in algae. Here, we identified OREs possibly localized to organelles in the unicellular rhodophyte Cyanidioschyzon merolae. We then examined intracellular localization of green fluorescent protein-fusion proteins of these enzymes in C. merolae, whose cell has a single plastid and a single mitochondrion and is suitable for localization analysis, demonstrating that the plastid and the mitochondrion contain markedly different components of replication machinery. Phylogenetic analyses revealed that the organelle replication apparatus was composed of enzymes of various different origins, such as proteobacterial, cyanobacterial, and eukaryotic, in both red algae and green plants. Especially in the red alga, many enzymes of cyanobacterial origin remained. Finally, on the basis of the results of localization and phylogenetic analyses, we propose a model on the succession of OREs in eukaryotes.

Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize

Chloroplasts and other members of the plastid organelle family contain a small genome of bacterial ancestry. Young chloroplasts contain hundreds of genome copies, but the functional significance of this high genome copy number has been unclear. We describe molecular phenotypes associated with mutations in a nuclear gene in maize (Zea mays), white2 (w2), encoding a predicted organellar DNA polymerase. Weak and strong mutant alleles cause a moderate (approximately 5-fold) and severe (approximately 100-fold) decrease in plastid DNA copy number, respectively, as assayed by quantitative PCR and Southern-blot hybridization of leaf DNA. Both alleles condition a decrease in most chloroplast RNAs, with the magnitude of the RNA deficiencies roughly paralleling that of the DNA deficiency. However, some RNAs are more sensitive to a decrease in genome copy number than others. The rpoB messenger RNA (mRNA) exhibited a unique response, accumulating to dramatically elevated levels in response to a moderate reduction in plastid DNA. Subunits of photosynthetic enzyme complexes were reduced more severely than were plastid mRNAs, possibly because of impaired translation resulting from limiting ribosomal RNA, transfer RNA, and ribosomal protein mRNA. These results indicate that chloroplast genome copy number is a limiting factor for the expression of a subset of chloroplast genes in maize. Whereas in Arabidopsis (Arabidopsis thaliana) a pair of orthologous genes function redundantly to catalyze DNA replication in both mitochondria and chloroplasts, the w2 gene is responsible for virtually all chloroplast DNA replication in maize. Mitochondrial DNA copy number was reduced approximately 2-fold in mutants harboring strong w2 alleles, suggesting that w2 also contributes to mitochondrial DNA replication.

Conservation of POPs, the Plant Organellar DNA Polymerases, in Eukaryotes

POPs, plant organellar DNA polymerases, have been isolated from various photosynthetic eukaryotes. Previously, we purified the native POP of Cyanidioschyzon merolae(CmPOP) from whole cellular extracts and showed that CmPOP has DNA polymerase activity with a high processivity and a 3'-5' exonuclease activity, and its expression is related to cell proliferation. In rice, the recombinant protein of POP has activities found in CmPOP, and high fidelity of POP has also been demonstrated. These facts suggest that POPs are involved in the replication of organellar genomes. POPs are also conserved in most non-opisthokont eukaryotes, which lack DNA polymerase γ (Polγ), a mitochondrial replication enzyme in opisthokonts (fungi and animals). The ciliateTetrahymena thermophilacontains a single gene for a putative POP (TetPOP). Immunoblot analysis demonstrated that TetPOP is localized in mitochondria, and TetPOP has been purified from mitochondria through a column chromatography series. Sensitivity to phosphonoacetate and dideoxyTTP was examined in POPs (TetPOP and CmPOP) or POP-containing organelles (chloroplasts of Arabidopsis) and other polymerases (DNA polymerase I and mitochondria of rat liver, which contain Polγ), and the results suggest that high sensitivity to phosphonoacetate is unique to POPs in Family-A DNA polymerases. Finally, we propose a model for the succession of organellar DNA polymerases.

Participation of Leaky Ribosome Scanning in Protein Dual Targeting by Alternative Translation Initiation in Higher Plants

Postendosymbiotic evolution has given rise to proteins that are multiply targeted within the cell. Various mechanisms have been identified to permit the expression of proteins encoding distinct N termini from a single gene. One mechanism involves alternative translation initiation (aTI). We previously showed evidence of aTI activity within the Arabidopsis thaliana organellar DNA polymerase gene POLgamma2. Translation initiates at four distinct sites within this gene, two non-AUG, to produce distinct plastid and mitochondrially targeted forms of the protein. To understand the regulation of aTI in higher plants, we used Polgamma2 as a model to investigate both cis- and trans-acting features of the process. Here, we show that aTI in Polgamma2 and other plant genes involves ribosome scanning dependent on sequence context at the multiple initiation sites to condition specific binding of at least one trans-acting factor essential for site recognition. Multiple active translation initiation sites appear to operate in several plant genes, often to expand protein targeting. In plants, where the mitochondrion and plastid must share a considerable portion of their proteomes and coordinate their functions, leaky ribosome scanning behavior provides adaptive advantage in the evolution of protein dual targeting and translational regulation.

Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.

Identification of essential genes for linear replication of an SCP1 composite plasmid

The 365 023 bp linear plasmid Streptomyces coelicolor plasmid 1 (SCP1) of S. coelicolor A3(2) belongs to the model systems of actinomycetes linear plasmids. Although the plasmid was detected more than two decades ago, little functional information about replication and maintenance of the linear topology exists. Here, for the first time, is the description of a region in SCP1 that is essential for linear replication of the plasmid. The SCP1 linear replication (Slr) region contains three putative genes. Intriguingly, one of the genes, SlrC, does show homology to PPA1294 of Propionibacterium acne that possesses a conserved DNA_pol_B_2 domain, which is found in organellar and viral DNA polymerases as, for example, in the Bacillus phi29 polymerase.