Which organelle replicates dna

As mentioned previously, Twinkle is similar to T7 gp4 protein and possesses both DNA helicase and primase activities. Nonetheless, plants do contain organellar RNA polymerases that could complement the activity of Twinkle [ 78 , 79 ]. Twinkle uses a unique recognition sequence to begin ribonucleotide synthesis and appears to prefer cytosine and guanine incorporation over uracil and adenine [ 99 ].

If either of the cryptic nucleotides or the guanine directly upstream from them are substituted, RNA synthesis is abolished. This is unique from other DNA primases, in that two cryptic nucleotides are required for synthesis whereas other primases often require one. The exact mechanism of Twinkle association with template DNA is not fully understood.

One theory points to Aquifex aeolicus , a primitive thermophilic bacteria, in which primer synthesis is initiated from a trinucleotide sequence composed of cytosines and guanines much like Arabidopsis Twinkle [ ]. This G—C rich sequence is hypothesized to provide stability during primer extension.

The co-evolution of nuclear, plastid, and mitochondrial genomes in plants has led to an interesting arrangement of RNA polymerases RNAP in the organelles. Unlike animal mitochondria, which utilize a single RNA polymerase [ ], plant organelles require multiple RNAPs: at least two for plastids and one for mitochondria.

Different species may possess multiple copies of these nuclear encoded organellar proteins, but the earliest phylogenetic versions of these enzymes exist in the waterlily Nuphar advena , a basal angiosperm [ ]. Extensive research has been performed on how plant RNA polymerases recognize promoters and transcribe genes. Three single-subunit mitochondrial RpoT genes have been identified in Arabidopsis ; however, only two have been proven to localize to mitochondria [ 79 , ].

A duplication of one of these genes has led to the creation of a RpoTmp. How these enzymes coordinate synthesis of RNA is largely unexplored, although some research suggests RpoTmp is responsible for gene synthesis in early seedling development and RpoTm and RpoTp take over once the plant has fully developed [ ].

NEP isolated from P. The enzyme was also found to be resistant to inhibition by tagetitoxin, a specific inhibitor of chloroplast-encoded RNA polymerase, as well as polyclonal antibodies specific to purified pea chloroplast RNAP. These findings suggest that plastids and probably mitochondria possess an RNAP gene that functions as a DNA primase, although further research on this topic is needed.

Unlike the mitochondrial phage-like RNA polymerases, the PEP is made up of multiple subunits that share homology with the core subunits of E. These subunits are encoded from the genes rpoA , rpoB , rpoC1 , and rpoC2 , respectively. In addition, several nuclear-encoded sigma factors form the PEP holoenzyme and provide promoter recognition for the plastid encoded subunits [ 76 ].

In agreement with the theory of endosymbiosis, the core enzyme of the NEP is also homologous to multi subunit RNA polymerases of cyanobacteria [ ]. This makes sense when observing that Twinkle, a helicase-primase, is present in animals but lacks the primase activity observed in both phage and plants. Unfortunately, the ability and scale on which this actually happens is grossly understudied, most likely due to the assumption that organellar DNA is primed by mimicking the simple replisome found in T7 phage.

Therefore, the ability of RNA polymerase to prime DNA for synthesis may be extremely important to plants and could be a fruitful area of research. Recent work has shown RNase H-like activity both in mitochondria and chloroplasts [ 80 ].

The first one is similar to bacterial SSBs. This protein has been shown to localize to both mitochondria and chloroplasts, and stimulates bacterial RecA activity [ 39 , 44 , 81 ]. RecA is a bacterial protein involved in homologous recombination and strand invasion, and is discussed in greater detail below. Although the function of these molecules has not been completely detailed, mutants for OSB1 accumulate mtDNA homologous recombination products.

In subsequent generations, these products segregate into separate plant lines where one of the homologous recombination products is predominant. If OSB1 activity is restored the plants segregate into separate line they will revert to wild type configurations of mtDNA. However, if segregation has already occurred, restoration of OSB1 activity does not restore plants to wild type mtDNA configurations [ 82 ]. Therefore, OSB proteins are most likely involved in recombination surveillance and preventing transmission of incorrect recombination products to newly formed mitochondria.

WHY proteins form tetramers that take on the appearance of a whirligig, hence the name Whirly. Whirly appears to have expanded roles from OSB proteins. Some WHY proteins have been associated with double stranded DNA repair [ 84 , 85 ] and regulation of defense genes in response to pathogens [ ]. In addition, plant organelles also contain proteins involved in homologous DNA recombination.

There are two classes of proteins dedicated to recombination in plant organelles. MutS from E. Due to this activity, this gene was originally called chm for chloroplast mutator.

Later, it was discovered that chm mutants cause rearrangements to the mitochondrial genome that lead to the observed phenotypes and defective chloroplasts. Despite extensive searching, no mutation or rearrangement of the plastid genome has been observed in chm mutants [ ].

Being homologous to MutS from E. Insertion mutations of yeast msh1 lead to a petite phenotype indicative of mitochondrial dysfunction. Mutation of yeast msh1 is also accompanied by large-scale point mutations and rearrangements in the mitochondrial genome [ ].

Interestingly, plant MSH1 mutants do not accumulate point mutations over time, suggesting that the plant MSH1 specializes primarily in recombination and is not essential for correcting mismatches. Instead, plant MSH1 possesses three recognizable domains and three unknown domains to facilitate mismatch repair. Point mutations to the ATPase and endonuclease domains of plant MSH1 led to the defective chloroplast phenotype [ ], suggesting that plant MSH1 may provide mismatch recognition and base excision without the need for MutL or MutH homologs, although this has not been experimentally shown.

Recent studies have shown that MSH1 suppresses homeologous recombination [ , ]. Plant MSH1 also has a unique GIY-YIG endonuclease domain, which binds specifically to the D-loop structure, suggesting that this protein may recognize and resolve mismatch-containing intermediates [ ]. RecA facilitates homologous recombination by correctly pairing homologous sequences and promoting strand invasion.

Eukaryote versions of this protein are called RAD All homologous recombination begins with strand invasion mediated by RecA family proteins, making this protein crucial for this type of repair. RecA functions by coating single stranded DNA at lesions to form presynaptic filaments. This complex will then search for homology within intact double stranded DNA. Once homology has been established, the presynaptic complex will destabilize the double stranded DNA promoting strand exchange and D-loop formation.

The rearrangements observed in RecA3 mutants are due to homologous recombination of repeated sequences in the mitochondrial genome. Reintroducing RecA3 into these mutants results in a reversal of this effect in most of the progeny by abolishing aberrant mitochondrial DNA molecules. RecA1 and RecA2 appear to be even more essential to homologous recombination as mutations in these genes cause a seed-lethal phenotype.

This may be explained by the lack of the C-terminal domain found in both RecA1 and RecA2 as well as bacterial homologs. In bacteria, deletion of this C-terminal domain enhances the activity of RecA, suggesting its involvement in autoregulation. The amount of information surrounding plant organellar DNA ligases is extremely limited.

Unlike mitochondria, no DNA ligase has been confirmed or observed functioning in plastids, representing a potential avenue of research, as the activity of this enzyme in both organelles must be present. Plant LIG1 knockouts are seedling-lethal and knockdown mutants display severe growth defects due to effects on the nuclear genome rather than the mitochondrial genome [ ].

Genomes found in plant mitochondria and chloroplasts are essential for organelle function, but there is still relatively little known about how these genomes are replicated and maintained. This is especially true for plant mitochondria, which have a very large variation in genome size depending on the species, and there is considerable evidence that the genome exists as linear subgenomic molecules, raising questions as to how the integrity of the genetic information is maintained.

Thus, plant mitochondrial genomes and their replication are much more complex than their animal counterparts. It is clear that for at least some replication functions more than one gene is present in Arabidopsis , suggesting the possibility of functional redundancy.

For chloroplasts, although a DNA replication mechanism has been established, it is quite possible that more than one mechanism is involved, perhaps for different stages of growth or in response to different signals. Further research is needed to better understand the basic structure of the organelle genomes and how these DNA molecules are replicated. In addition, the mechanism s for maintaining genome copy number and regulation of replication initiation are not known and should be studied.

We acknowledge the assistance of several undergraduate students on various projects in the BLN research laboratory related to plant mitochondrial DNA replication and recombination. Conceptualization of the ideas for this review were by S. Writing, original draft preparation including figures and tables were by S.

Funding acquisition, B. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. National Center for Biotechnology Information , U. Journal List Plants Basel v.

Plants Basel. Published online Sep Stewart A. Brent L. Author information Article notes Copyright and License information Disclaimer. Louis, Mo , USA. Received Aug 13; Accepted Sep This article has been cited by other articles in PMC. Abstract 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.

Introduction 1. Discovery of Mitochondria and Chloroplasts In Robert Hooke became the first person to observe cells with a simple microscope [ 1 ]. Evolutionary Origins of Each Organelle Both mitochondria and chloroplasts are believed to have originated through endosymbiosis.

Organelle Genomes and Structure 2. Genome Size Endosymbiosis is accompanied with massive gene transfer to the nucleus of the host cell, resulting in considerable size reduction of the genome of the incoming cells. Open in a separate window. Figure 1. Genome Structure and Content In both mitochondria and chloroplasts the DNA is associated with positively charged proteins in nucleoids [ 12 ].

Organelle DNA Replication 3. Figure 2. Plant Mitochondria Plants most likely employ multiple mechanisms for replication of the mtDNA due to the complex structure of the mitochondrial genome.

Figure 3. Organelle DNA Replication Proteins The genomes in both mitochondria and chloroplasts are complexed with positively charged proteins in nucleoids [ 12 , 68 ], and this is the form of the DNA that is replicated in the organelles. Table 1 Proteins involved in plant organellar DNA replication. Bacterial P [ 68 ] EXO2? Primer Removal In E. Conclusions Genomes found in plant mitochondria and chloroplasts are essential for organelle function, but there is still relatively little known about how these genomes are replicated and maintained.

Acknowledgments We acknowledge the assistance of several undergraduate students on various projects in the BLN research laboratory related to plant mitochondrial DNA replication and recombination. Author Contributions Conceptualization of the ideas for this review were by S. Conflicts of Interest The authors declare no conflict of interest.

References 1. Hooke R. Science Heritage, Ltd. Harris H. The Birth of the Cell. Ernster L. Mitochondria—A Historical Review.

Cell Biol. Schimper A. Botanische Zeitung. Timmis J. Endosymbiotic gene transfer: Organelle genomes forge eukaryotic chromosomes. Kutschera U. Endosymbiosis, cell evolution, and speciation.

Theory Biosci. Zimorski V. Endosymbiotic theory for organelle origins. Bergthorsson U. Widespread horizontal transfer of mitochondrial genes in flowering plants. Blanchard J. Pervasive migration of organellar DNA to the nucleus in plants.

Boore J. Animal mitochondrial genomes. Nucleic Acids Res. Iborra F. The functional organization of mitochondrial genomes in human cells. BMC Biol. Kukat C. Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Montier L. Number matters: Control of mammalian mitochondrial DNA copy number.

Fauron C. Plant Mitochondrial Genomes. In: Daniell H. Springer; Dordrecht, The Netherlands: Oldenburg D. The amount and integrity of mt DNA in maize decline with development. Preuten T. Fewer genes than organelles: Extremely low and variable gene copy numbers in mitochondria of somatic plant cells.

Plant J. Daniell H. Chloroplast genomes: Diversity, evolution, and applications in genetic engineering. Genome Biol. Palmer J. Comparative Organization of Chloroplast Genomes. Kolodner R. The A. Additionally, the Atpop2 mutant displayed high sensitivity to ciprofloxacin, an inducer of DNA double-strand breaks. TWINKLE T7 gp4-like protein with intramitochondrial nucleoid localization , which is a homolog of the T7 phage gp4 protein with primase and helicase activities, was originally reported to function as a hexameric DNA helicase in human mitochondria Spelbrink et al.

Dual-targeted enzymes to the mitochondria and chloroplasts of plants are summarized in the review by Carrie and Small Red algae and diatoms have a plastid-encoded DnaB helicase and a nucleus-encoded DnaG primase.

We also confirmed the plastid-localization of DnaG in the red alga Porphyridium purpureum. In addition to gyrases, plant organelles contain A-type topoisomerase I, which is a homolog of bacterial topoisomerase I TopA.

To search for mitochondrial topoisomerases, we examined the subcellular localization of topoisomerases encoded in the Cyanidioschyzon merolae genome, and showed that a homolog of eukaryotic TOP2 is targeted to mitochondria. To date, organellar localization of eukaryotic TOP2 has not been reported in plants. In Cyanidioschyzon merolae , the gyrase specific inhibitor nalidixic acid arrests not only replication of the plastid genome, but also that of the mitochondrial and nuclear genomes Itoh et al.

The localization results of gyrases in Cyanidioschyzon merolae suggest that defective plastid replication leads to the arrest of mitochondrial and nuclear replication by a yet unknown mechanism. Four DNA ligases have been identified in the A. AtLIG1 is expressed in all tissues of A. However, it has been noted that AtLIG6 has a putative plastid-targeting peptide at the N -terminus and might therefore be targeted to plastids Sunderland et al.

Cyanidioschyzon merolae has a single gene encoding DNA ligase. Cyanidioschyzon merolae DNA ligase 1 CmLIG1 has two methionine residues in its N -terminal region and is targeted to both mitochondria and plastids when the transcript is translated from the first and second initiation codons Moriyama et al. Therefore, CmLIG1 appears to have triple localization in plastids, mitochondria, and the nucleus. AtSSB1 is localized to mitochondria, but was also reported to be localized to chloroplasts in the review by Cupp and Nielsen PDF motifs are conserved only in green plants, including Chlamydomonas reinhardtii.

In our analysis, the SSB of Cyanidioschyzon merolae is localized only in the mitochondrion, unlike that of A. We performed the localization analysis using a construct starting from the second methionine codon or starting from the ATA codon located upstream of the first methionine codon; however, none of the constructs showed plastid localization.

We also examined the organellar localization of RPAs in Cyanidioschyzon merolae , and even though they have no extension sequence at the N -terminus, they were localized to the nucleus. Based on these findings, the plastidial SSB in red algae remains unidentified. In contrast, RNaseH1 performs this role in human mitochondria Kasiviswanathan et al. Cyanidioschyzon merolae has a gene with high sequence homology to bacterial PolI Moriyama et al.

Phylogenetic analyses of bacterial-type replicative enzymes have been performed Moriyama et al. However, the analyses indicated that red algal DnaB helicase and DnaG primase originated from cyanobacteria Figure 3A. Gyrases A and B also originated from cyanobacteria in both green plants and red algae Figure 3B.

Phylogenetic trees of enzymes related to organellar genome replication. Simplified phylogenetic trees A—D. Modified from Moriyama et al. The enzymes related to organellar DNA replication and recombination in a species of angiosperm, fern, moss, filamentous terrestrial alga, two green algae, and two red algae are listed in Table 1. DnaB and DnaG are conserved only in red algae. The retention of SSBs is highly variable in photosynthetic eukaryotes. Bacterial-type SSB proteins are conserved in land plants and Cyanidioschyzon merolae , whereas OSB proteins are conserved among land plants, including A.

According to this classification, Physcomitrella patens and K. Conservation of origin-binding protein ODB is more limited, as only land plants have this protein. Therefore, all photosynthetic eukaryotes contain proteobacteria-derived PolI. The observed distribution of enzymes that play key roles in replication indicates that they are essentially conserved in all plants and algae. In contrast, because recombination-related enzymes and SSBs are non-uniformly distributed among plants and algae, these enzymes are considered to exhibit high plasticity during evolution.

TABLE 1. List of replication-related enzymes possibly localized to plastids or mitochondria in photosynthetic eukaryotes. Based on the presence of enzymes related to organellar genome replication in plant genomes, we propose a model for the substitution of these enzymes in photosynthetic eukaryotes Figure 4.

In red algae, most replication enzymes in the ancestor of photosynthetic eukaryotes are found in present-day species. Proposed model for the exchange of organellar replication enzymes during the evolution of photosynthetic eukaryotes. In the past decade, most enzymes related to plastid and mitochondrial DNA replication in plants and algae have been identified. These studies have revealed that the core enzymes and components involved replication are identical in the plastids and mitochondria of land plants.

In contrast, SSBs and recombination-related enzymes are not universally conserved in the green lineage, suggesting that these enzymes are possibly susceptible to exchange or loss during evolution, leading to the acquisition or creation of species-specific enzymes. Unlike the green lineage, red algae contain different replicative protein profiles in plastids and mitochondria. Red algal plastids contain numerous replication proteins that originated from cyanobacteria Moriyama et al.

To date, a number of organelle-localized enzymes have been identified. However, biochemical data are lacking for the majority of organellar replication enzymes in plants. The role of an enzyme predicted by homology searches against known enzymes might differ from its actual function or properties. The regulatory mechanisms controlling the initiation of plant organellar genome replication and the number of organellar DNA copies remains to be explored. Recently, chloroplast DNA replication was shown to be regulated by the cellular redox state in the green alga Chlamydomonas reinhardtii Kabeya and Miyagishima, Specifically, chloroplast DNA replication was activated and inactivated by the addition of reducing and oxidative agents, respectively, in both in vivo and in vitro assays.

Light-dependent genome replication was also reported in cyanobacteria, in which DCMU [3- 3,4-dichlorophenyl -1,1-dimethylurea], an inhibitor of electron transport between the PSII complex and plastoquinone pool, inhibits DNA replication initiation, and DBMIB 2,5-dibromoisopropylmethyl-p-benzoquinone , an inhibitor of electron transport between plastoquinone and cytochrome b 6 f complex, inhibits the initiation and elongation of replication Watanabe et al.

Thus, the light-mediated replication of plastid DNA in algae may have originated from cyanobacteria. However, organellar replication in land plants and multicellular plants appears to be regulated by other mechanisms. In land plants, the replication of organellar genomes is restricted to meristematic tissues, and is not associated with the cycle or organellar division Hashimoto and Possingham, ; Fujie et al.

These findings suggest that land plants have more complex regulatory mechanisms controlling the replication of organellar genomes than those operating in algae. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Arnold, J. Human mitochondrial RNA polymerase: structure-function, mechanism and inhibition. Acta , — Carrie, C.

Approaches to defining dual-targeted proteins in Arabidopsis. Plant J. A reevaluation of dual-targeting of proteins to mitochondria and chloroplasts. Chavalitshewinkoon-Petmitr, P. Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum. Cho, H. DNA gyrase is involved in chloroplast nucleoid partitioning. Plant Cell 16, — Christensen, A.

Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons. Plant Cell 17, — Cupp, J. Minireview: DNA replication in plant mitochondria. Mitochondrion doi: Diray-Arce, J.

D, Hunt, T. BMC Plant Biol. Mutated residues were selected based off of DCA analysis results. While only two mutations caused a disruption of Twinkle with Pol1A, most mutations weakened or completely disrupted interaction with Pol1B. Mutations in Pol1A were much more distinct, either completely disrupting interaction with Twinkle, or failing to affect this interaction at all.

To make Twinkle truncations we designed primers that shortened the protein in 10 amino acid increments from either the N-terminal or C-terminal side of the interacting region. We amplified and tested different truncations until the interaction was lost.

After finding the maximum truncation from both the N-terminal and C-terminal regions that retained a positive interaction, we created a final truncation that combined these borders. Residues — of Twinkle make up the smallest truncation that maintains interaction with Pol1A or Pol1B, as illustrated in Fig. This region does not reside in any predicted functional domain and further strengthens our hypothesis that the N-terminal region of Twinkle coordinates interaction with DNA polymerase.

Design and results of Twinkle protein truncations. Each line represents a 10 amino acid truncation from either the N-terminal or C-terminal side of Twinkle.

We next truncated the DNA polymerase genes as we did with Twinkle in order to hone in on a specific interacting region. To do this we used the minimal region identified from the Twinkle truncations as a binding partner. Before designing primers for the 10 amino acid deletions, we produced truncations using the primers originally designed in our library approach Fig. The purpose was to identify a significantly smaller region of the DNA polymerases that would still interact with Twinkle in order to reduce the amount of effort required to produce dozens of 10 amino acid truncations.

However, we found that we were unable reduce the interacting region of the polymerases much further than our initial screen had revealed Fig.

We also noticed that different regions of Pol1A and Pol1B were able to interact with the small region of Twinkle we had identified. Spot dilutions of each interaction allude to a much stronger association between Pol1A and Twinkle versus Pol1B with Twinkle.

Truncations are numbered 1—12 and span Pol1A and Pol1B as designated above. Each truncation was tested for association with the interacting region of Twinkle and plated in spot dilutions on selective and non-selective media as a control. Non-selective media is SD -leu -trp and the selective media is SD -leu -trp -his -ade. Thermophoresis is a biochemical assay that measures interaction between molecules through the use of a temperature gradient.

Molecules, in this case proteins, are suspended in liquid which is heated by a laser. The laser is focused to heat a specific point of the liquid, creating a temperature gradient in the surrounding area. Movement of molecules through this temperature gradient indicates the relative size of the molecules with smaller molecules moving quicker than larger ones.

If two molecules or proteins interact with each other, the larger size of the complex retards its movement through the liquid. This movement can be used to calculate dissociation constants of two interacting molecules. Using thermophoresis we were able to determine the binding strength between Twinkle and different regions of Pol1B.

After incubation with increasing concentrations of ligand, samples were loaded into MST standard capillaries. Pol1B showed specific binding to full length Twinkle, the primase and helicase domains, and the zinc finger subdomain. The fitted values from the thermophoretic analysis yielded dissociation constants for the ligand partners Pol1B-Twinkle of 1. These values are comparable with the binding affinities between T7 DNA polymerase and the bifunctional T7 primase-helicase [ 22 , 23 , 24 ].

The dissociation constants for this interaction are in the order of 0. In Bacillus subtilis the dnaG primase protein has been documented to interact with the DNA polymerase dnaE helicase with a dissociation constant of 0. Finally, zinc finger containing proteins have been described as interacting partners with DNA polymerases, in which cases the zinc finger is critical for physical interaction between both proteins [ 27 , 28 ]. The confirmation of the interaction between the Twinkle and Pol1B provide further support for the role of these enzymes in replicating the Arabidopsis thaliana organellar genome.

The thermophoretic data was fitted to the Kd equation described in materials and methods. The zinc finger subdomain was able to bind to the PolB and a Kd was measured at 2. The RNAP subdomain red did not show binding data not shown. The primase and helicase domain were able interact with the polymerase with a Kd of 0. The full-length protein interacts with a Kd of 1.

All proteins were titrated in 16 serial dilutions from different concentrations. Graphs were plotted at x axis with enzyme concentration and at the y axis with normalized fluorescence. Error bars represent the standard error for three measurements. Surprisingly, the Twinkle mutants appear unaffected by the loss of this protein Fig.

This was followed with relative qPCR analysis of genome copy numbers, which showed no significant change in mitochondrial or chloroplast DNA levels relative to the nuclear genome due to the Twinkle or PrimPol insertion mutations Fig. Considering that Twinkle homologs are essential for both T7 phage and animal mitochondria genome maintenance, we were surprised to see no apparent phenotype or genome copy number changes in the mutant plants compared to wild-type plants.

Growth and genome copy number differences between WT and Twinkle mutant Arabidopsis plants. The y-axis represents a percentage of the genome copy numbers present in mitochondria and chloroplasts relative to WT plants. Within this region, mutation of several key residues results in complete dissociation from Pol1A or Pol1B as determined by yeast-two-hybrid analysis.

We have also shown that mutation of key residues in Pol1A disrupts interaction with Twinkle. While the key residues in Twinkle are fairly closely clustered, these key residues in the DNA polymerases are spaced much further apart. We suspect this was the main reason we could not produce a smaller truncation of Pol1A or Pol1B that maintained interaction with Twinkle, whereas the interacting region of Twinkle was localized to a very narrow region of the protein.

However, it does not confirm that only the N-terminal region of the DNA polymerases is crucial for positive interaction with Twinkle. Results from our previous screens also show this same region of Twinkle associates with SSB1, supporting the idea that Twinkle coordinates the assembly of a minimal DNA replisome.

As most vascular plants possess the same orthologs of these organellar replication proteins, this pattern is likely to be repeated in higher plant chloroplasts and mitochondria of other species. Although we have provided evidence showing that Twinkle and the DNA polymerases likely form a minimal DNA replisome, we do not know if this is the sole DNA replisome utilized in plant organelles. Additionally, other proteins may provide accessory functions such as in E.

For example, almost no research has been performed on primer removal. Ribonucleases have been reported in plant mitochondria and chloroplasts, but their characterized functions are in mRNA and tRNA processing [ 33 ]. Pol1A and Pol1B have been shown to be processive enough to replicate an entire organelle genome equivalent [ 17 , 34 ]. This is much more processive than E. Interestingly, recombinant versions of E. Plant organelle DNA polymerases may also bind thioredoxin or other processivity factor to achieve their greater processivity, but this has never been shown.

If this occurs, it would help explain why these enzymes possess much greater processivity than E. We have demonstrated an association between the DNA polymerases and Twinkle suggesting that these two proteins are part of the DNA replisome in these organelles.

However, homozygous Twinkle T-DNA insertion mutants in Arabidopsis show no noticeable defect in plant growth and there is no noticeable decrease in organelle genome copy number compared to wild-type plants Fig.

The plants grow similarly to WT. This is puzzling, as similar interactions between T7 gp4 helicase-primase are essential for processive replication of phage DNA [ 24 ].

In addition, conditional Twinkle knockout mice fail to survive and display a rapid depletion of mitochondrial DNA in both heart and skeletal muscle tissue [ 37 ]. If Twinkle knockout plants grow well, this strongly suggests that another protein may provide the primase activity required for DNA polymerases to function. This same study demonstrated that Arabidopsis Twinkle cannot prime T7 or E.

We and others Brieba and Morley, unpublished observations have shown that plants can survive with almost no visible growth defects if they have at least one functional organellar DNA polymerase. Growth at these decreased organelle genome copy numbers does not appear to be affected by low light conditions or drought data not shown.

In either case, there is an apparent redundancy in Arabidopsis for Twinkle function. However, T-DNA insertion mutations in either of these proteins also lead to no discernable growth defect unpublished observations. In any case, Pol1A and Pol1B must be involved, since they are the only DNA polymerases found in the organelles and we and others have been unable to make double homozygous mutants in both genes. In previous studies, we reported that plants with Pol1B mutations had a greater decrease in organelle genome copy number relative to plants with Pol1A mutations, particularly in mitochondria [ 38 , 39 ].

We also observed a slight delay in growth of these plants. If the minimal replisome consists of Twinkle and a DNA polymerase, we expected based on our previous results that Twinkle would show a strong association with Pol1B. However, our research shows a stronger association with Pol1A. This could be explained by different scenarios: a strong interaction with Twinkle may be detrimental to DNA replication, or the roles of Pol1A and Pol1B may be less redundant and more distinct from each other.

Other studies showed that both DNA polymerases can perform translesion repair but Pol1B is more effective at strand displacement than Pol1A [ 19 , 40 ]. This contrasts with findings in maize, where only one of the two organellar DNA polymerases was shown to be responsible for replication of the maize plastid genome, as mutation of this single gene essentially abolished chloroplast DNA replication [ 41 ].

This maize mutant had about a two-fold reduction in mitochondrial DNA, implying that the second DNA polymerase may function in mitochondria. In addition, recombination dependent replication RDR could explain the lack of a phenotype in Twinkle mutants. Extensive use of RDR in plant organelles has been demonstrated as a means of maintenance and repair of mitochondrial and plastid DNA [ 42 ]. Mutations in recombination proteins lead to genome instability and are often lethal.

One prominent example is RecA, a homolog of the bacterial replication protein RecA. Mutations to these homologs lead to delayed phenotypes, increased recombination, and in the case of RecA2 plant death beyond the seedling stage [ 43 , 44 , 45 ].

Mutations to any of these proteins lead to an increase in illegitimate recombination and adversely affect plant development. We have used a classic molecular biology technique yeast-two-hybrid , bioinformatics DCA , and biochemistry thermophoresis to define important regions and residues that are key to these interactions.

This three pronged approach provides confirmation of the interactions, and can be applied to other protein-protein interaction studies. Further work examining the complete DNA replisome of plant organelles will identify other proteins involved in mitochondrial DNA replication in Arabidopsis , including when Twinkle is mutated.

The same approach we have used to demonstrate the interaction between Twinkle and the DNA polymerases may reveal specific regions in these candidate proteins that are crucial for DNA replisome assembly and function. We calculated both direct and indirect interactions between amino acid residues in the DNA polymerase PolIA, At1g and PolIB, At3g and Twinkle At1g genes through a direct coupling analysis of sequences from 90 plant species.

First, we manually downloaded each DNA polymerase and Twinkle gene from the National Center for Biotechnology Information NCBI [ 50 ], ensuring that each plant species included complete gene annotations for both genes. Following the individual alignments of each set of sequences, we joined the aligned sequences for each species into a single FASTA file by adding 20 asparagine residues to the end of the Polymerase II gene followed by concatenating the Twinkle sequence for each species.

This artificial buffer ensured that interactions identified between the two genes were not affected by combining the genes e. The output file contains four columns: the position of the first amino acid residue, the position of the compared amino acid residue, the amount of direct information between the two residues, and the amount of mutual information between the two residues.

Since the amino acid residues reported in the DCA analysis came from the combined multiple sequence alignments i. We then renumbered the amino acid residues to be congruent with the original unaligned sequences i. Finally, using these labels, we created a heatmap of the calculated mutual information using the matplotlib and scipy.

We used the heatmap to visually identify areas of higher mutual information between the two genes. Yeast-two-hybrid analysis was performed using materials and protocols from Clontech.

This included using the Matchmaker gold strain of yeast for small scale yeast transformations following the lithium acetate protocol outlined by Clontech.

These truncations were cloned first into E. Appropriate haploid yeast libraries were subsequently mated and plated on media selective for positive interactions. All constructs identified in positive interactions were tested in both bait and prey plasmids, and for autoactivation by transforming the target protein against an empty bait or prey plasmid to eliminate the possibility of false positive interactions. For mating experiments, Matchmaker gold yeast was used in conjunction with an Arabidopsis library of normalized cDNAs purchased from Clontech.

Tri-alanine substitution mutants were created to test the effects of mutating the residues highlighted by DCA analysis. The resulting PCR product was a plasmid sized blunt ended DNA molecule possessing the tri-alanine substitution we had designed. The blunt ends of this molecule were ligated together and transformed into E. Colonies were picked, grown and harvested for plasmid DNA which was checked for correct insertion of the tri-alanine substitution via Sanger sequencing.

Once verified, the plasmids were transformed into yeast and measured for interaction using selective media. Twinkle lacking the first 91 codons was used as template for the construction of the DNA primase and helicase domains, and the RNAP and zinc finger subdomains. The primase domain residues 92— , RNAP and zinc finger subdomains residues — and 92—, respectively were cloned into pETb and purified as described [ 12 , 19 ].

The helicase domain — was cloned into the pCri-1b vector [ 54 ] and purified as described before, changing Tris-HCl to potassium phosphate pH7. The ligands were titrated against labeled Pol1B in 16 serial dilutions from