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Encyclopedia of Family A DNA Polymerases Localized in Organelles: Evolutionary Contribution of Bacteria Including the Proto-Mitochondrion - PubMed

  • ️Mon Jan 01 2024

Encyclopedia of Family A DNA Polymerases Localized in Organelles: Evolutionary Contribution of Bacteria Including the Proto-Mitochondrion

Ryo Harada et al. Mol Biol Evol. 2024.

Abstract

DNA polymerases synthesize DNA from deoxyribonucleotides in a semiconservative manner and serve as the core of DNA replication and repair machinery. In eukaryotic cells, there are 2 genome-containing organelles, mitochondria, and plastids, which were derived from an alphaproteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNA polymerases that localize and work in them to maintain their genomes. The evolution of organellar DNA polymerases has yet to be fully understood because of 2 unsettled issues. First, the diversity of organellar DNA polymerases has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNA polymerases that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNA polymerases known to date show no phylogenetic affinity to those of the extant alphaproteobacteria or cyanobacteria. In this study, we identified from diverse eukaryotes 134 family A DNA polymerase sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNA polymerases were further examined experimentally. The results presented here suggest that the diversity of organellar DNA polymerases has been shaped by multiple transfers of the PolI gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed 2 mitochondrial DNA polymerases, POP, and a candidate of the direct descendant of the proto-mitochondrial DNA polymerase I, rdxPolA, identified in this study.

Keywords: DNA polymerase; endosymbiosis; last eukaryotic common ancestor; lateral gene transfer; mitochondria; plastids.

© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.

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Figures

Fig. 1.
Fig. 1.

Global phylogeny of family A famA DNAPs sampled from bacteria, phages, and eukaryotes. The ML tree was inferred from an alignment comprising 488 famA DNAP sequences with 355 aa positions. The terminal branches of the famA DNAPs originated from bacteria, phages, and eukaryotes are shown in different colors (the color-coding scheme is provided in the inset). The branches of 15 types of famA DNAP found in eukaryotes are shaded. We present the ultrafast bootstrap support (UFBP) value for the node uniting the paraphyletic chloroPolA sequences and cyanobacterial PolI sequences on the corresponding node. Otherwise, the UFBP value for the monophyly of each type of eukaryotic famA DNAP is shown in the corresponding shaded area.

Fig. 2.
Fig. 2.

Subcellular localization of GFP fused with the N-terminus of chlnmPolA when expressed in chlorarachniophyte cells. GFP fused with the first 100 amino acid residues of the chlorarachniophyte Bigelowiella natans chlnmPolA was expressed in the chlorarachniophyte Amorphochlora amoebiformis. The red color corresponds to chlorophyll autofluorescence. The green signal represents the GFP localization. The GFP signal was found to be associated tightly with but distinct from plastids, suggesting the protein is localized in the PPC. The scale bars are 10 µm.

Fig. 3.
Fig. 3.

Subcellular localizations of GFP fused with the N-termini of the 3 organellar family A DNAPs found in the cryptophyte Guillardia theta when expressed in diatom cells. GFP fused with the N-terminal 332 amino acid residues of G. theta cryotoPolA a), 100 residues of G. theta POP1 b and c), and 516 residues of G. theta POP2 d and e) were heterogeneously expressed in the diatom Phaeodactylum tricornutum UTEX642. In a, c, d, and e, the red color corresponds to chlorophyll autofluorescence and the green signal represents GFP. In b, the red color indicates both chlorophyll autofluorescence and mitochondria stained by MitoTracker Orange, and the green signal represents GFP. The scale bars are 10 µm.

Fig. 4.
Fig. 4.

Subcellular localizations of GFP fused with the N-termini of 5 rdxPolAs when expressed in yeast cells. GFP fused with the N-terminal amino acid residues predicted as the MTS of rdxPolA proteins from 3 members of Discoba (Ophirina amphinema, Tsukubamonas globosa, and Naegleria gruberi; a to c), the malawimonad (Gefionella okellyi; d), and the ancyromonad (Fabomonas tropica; e). The green signal corresponds to GFP. The red color indicates mitochondria stained by MitoTracker Red. The results presented here demonstrate that the mitochondrial translocons in yeast recognize the N-termini of the 5 rdxPolAs as the MTS. The scale bars are 5 µm.

Fig. 5.
Fig. 5.

Phylogenies that clarified the origins of 6 novel types of family A DNAPs localized in organelles. The phylogenies assessing the origins of rgPolA (147 sequences, 781 aa positions; panel a), chloroPolA (192 sequences 401 aa positions; panel b), pyramiPolA and eugPolA (161 sequences, 893 aa positions; panel c), and rdxPolA and cryptoPolA (229 sequences, 782 aa positions; panel d). The trees shown here were inferred with the ML phylogenetic method. ML nonparametric bootstrap support values are presented only for nodes key to the origins of the DNAPs of interest. In addition, the key nodes that received Bayesian posterior probabilities equal to or greater than 0.95 are marked by dots. The clades/branches are color-coded, as shown in the inset. In panel b), the PolI of the cyanobacterium Gloeomargarita lithophora is marked with a allow.

Fig. 6.
Fig. 6.

Summary of organellar family A DNAPs and their phylogenetic distributions. This figure summarizes the diversity of organellar family A DNAPs and their phylogenetic distributions over the tree of eukaryotes (schematically shown on the left). The presence of each type of organellar DNAP is indicated by a dot colored in blue (mitochondrion-localized; MT), red (PL), or green (nucleomorph-localized; NM), respectively. pyramiPolA found in green algae of the order Pyramimonadales is shown in a gray dot, as its exact organellar (mitochondrial or plastid) localization remains inconclusive. None of the currently known members of Breviatea or Metamonada, which bear highly reduced mitochondria whose own genomes had been discarded completely, is anticipated to retain any organellar DNAP, and the corresponding rows were left blank. Although we are aware of the laterally acquired POP in a subgroup of choanoflagellates (see supplementary fig. S8, Supplementary Material online), we regard the choanoflagellate POP as an exception and omitted it from this figure. We found no candidate for mitochondrial DNAP in the transcriptomes of hemimastigophorans and thus commented (“NO DATA AVAILABLE”).

Fig. 7.
Fig. 7.

Phylogenomic analyses of eukaryotes. a) The phylogenetic relationship among 97 eukaryotes was inferred from a phylogenomic alignment comprising 340 proteins by using the ML method. Major clades are shown by triangles. Dots on nodes indicate that the corresponding bipartitions receive UFBP values of 100%. The assemblage/lineages/species that use rdxPolA as mitochondrion-localized DNA polymerase are marked by stars. b) Alternative topologies of the eukaryotic phylogeny assessed in an AU test. We here examined only the phylogenetic relationship among 6 major clades of eukaryotes, namely Diaphoretickes (Diap), Amorphea plus CRuMs (A + C), Ancyromonadida (Ancy), Malawimonadida (Mala), Discoba (Disc), and Metamonada (Meta). Trees 2 to 7 were identical to the ML tree topology (Tree 1), except for the position of Amorphea plus CRuMs. The internal relationship in each major clade (see above) was re-optimized. Tree 8 and 9 are the best trees searched under the topological constraints assuming the monophyly of the POP-bearing and the rdxPolA-bearing lineages, respectively. The dotted lines in Trees 8 and 9 represent the constraints during the tree search described above. The rdxPolA-bearing lineages are marked by stars. For each test tree (Trees 2 to 9), the difference in log-likelihood from the ML tree (ΔlogL) and the P-value from the AU test are listed.

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