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, 430 (5), 737-750

Novel Families of Archaeo-Eukaryotic Primases Associated With Mobile Genetic Elements of Bacteria and Archaea

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Novel Families of Archaeo-Eukaryotic Primases Associated With Mobile Genetic Elements of Bacteria and Archaea

Darius Kazlauskas et al. J Mol Biol.

Abstract

Cellular organisms in different domains of life employ structurally unrelated, non-homologous DNA primases for synthesis of a primer for DNA replication. Archaea and eukaryotes encode enzymes of the archaeo-eukaryotic primase (AEP) superfamily, whereas bacteria uniformly use primases of the DnaG family. However, AEP genes are widespread in bacterial genomes raising questions regarding their provenance and function. Here, using an archaeal primase-polymerase PolpTN2 encoded by pTN2 plasmid as a seed for sequence similarity searches, we recovered over 800 AEP homologs from bacteria belonging to 12 highly diverse phyla. These sequences formed a supergroup, PrimPol-PV1, and could be classified into five novel AEP families which are characterized by a conserved motif containing an arginine residue likely to be involved in nucleotide binding. Functional assays confirm the essentiality of this motif for catalytic activity of the PolpTN2 primase-polymerase. Further analyses showed that bacterial AEPs display a range of domain organizations and uncovered several candidates for novel families of helicases. Furthermore, sequence and structure comparisons suggest that PriCT-1 and PriCT-2 domains frequently fused to the AEP domains are related to each other as well as to the non-catalytic, large subunit of archaeal and eukaryotic primases, and to the recently discovered PriX subunit of archaeal primases. Finally, genomic neighborhood analysis indicates that the identified AEPs encoded in bacterial genomes are nearly exclusively associated with highly diverse integrated mobile genetic elements, including integrative conjugative plasmids and prophages.

Keywords: DNA replication; Thermococcus plasmids; evolution; helicases; structural modeling.

Figures

Image 1
Fig. 1
Fig. 1
Global diversity of AEP proteins. Protein sequences were clustered by the pairwise sequence similarity (CLANS P-value ≤ 1e − 11). Representative members of well-characterized AEP groups are shown in green; PrimPol-PV1, red. Zoom-in on the PrimPol-PV1 group is shown in Fig. S1.
Fig. 2
Fig. 2
Comparison of conserved motifs across different AEP families. (A) Alignment of sequence logos from PolpTN2 homolog families and PriS from archaea and eukaryotes. Residues are colored by their chemical properties (polar, green; basic, blue; acidic, red; hydrophobic, black; neutral, purple). Residues thought to be involved in nucleotide binding in PolpTN2 are starred. (B) Active sites of PolpTN2 and human primase. Model of PolpTN2 is aligned with the structure of human primase (PDB: 4bpw). Metal ions are shown as yellow spheres. Incoming UTP is shown in cyan. (C) Covariation of residues in the PrimPol domain of PrimPol-PV1 and PriS homologs. The data set (1427 sequences) was collected by running 10 Jackhmmer iterations against nr70 database. Note that His residue at position 179 is primarily contributed by PriS homologs. PSICOV covariation score is given near the edges of triangle. Thickness of the line is proportional to the covariation score. WebLogos for each position are shown at the tips.
Fig. 3
Fig. 3
Catalytic activities of the wt and mutant PolpTN2 proteins. (A) The M1 and R231A mutants of PolpTN2 lost the DNA polymerase activity. The wild-type (lane 2) and two mutant forms of the recombinant protein PolpTN2 (lanes 3 and 4) or Taq polymerase (lane 5) were incubated with a short primer-template substrate (5-32P-labeled 20-nt oligonucleotide hybridized to 42-nt template). In the control reaction with no protein added (lane 1), no primer elongation was observed. (B) The M1 and R231A mutants of PolpTN2 lost the dNTP-dependent primase/polymerase activity. Primase reactions were performed using 42-nt-long oligonucleotide template blocked at 3′ end with the 3′-Spacer C3 (indicated with 3 Cs). Lane 1, wt PolpTN2; lane 2, mutant M1; lane 3, mutant R231A; lane 4, Taq DNA polymerase; lanes 5 and 6, labeled length markers. The variation in the length of synthesized products in lane 1 could be due to the terminal transferase activity of the PolpTN2 .
Fig. 4
Fig. 4
Taxonomic distribution of bacterial AEP of the PrimPol-PV1 supergroup. Taxa containing less than 4% (10% for RepB′) of representatives were merged into group “others.” A more detailed composition of each PrimPol-PV1 family is provided in Supplementary data file 1. Taxonomic distribution of bacterial AEP of the PrimPol-PV1 supergroup. Taxa containing less than 4% (10% for RepB′) of representatives were merged into group “others.” A more detailed composition of each PrimPol-PV1 family is provided in Supplementary data file 1.
Fig. 5
Fig. 5
Selected genome maps representing the diversity of MGEs encoding AEPs from the seven families of the PrimPol-PV1 supergroup. Each family is represented by one genome map. The maps are drawn roughly to scale. The protein-coding genes are shown by arrows indicating the direction of transcription, whereas tRNA genes are depicted by red bars. The blank arrows show poorly conserved genes, many of them encoding uncharacterized proteins. AEPs from different families are shown in red; genes encoding mobilization proteins are in cyan; integrase genes (Int) are in green, and genes encoding proteins responsible for virion assembly are depicted in yellow. Abbreviations: SF1 and SF2 hel, helicases of superfamilies 1 and 2, respectively; wHTH, winged HTH; TPR, tetratricopeptide repeat-containing protein; tr. Reg., transcription regulator; RT, reverse transcriptase; Topo IA, topoisomerase IA; MTase, DNA methyltransferase; REase, restriction endonuclease; TPase, transposase; TMP, tape measure protein; MCP, major capsid protein; TerS and TerL, small and large subunits of the terminase complex, respectively; SMC_N, N-terminal ATPase domain of the structural maintenance of chromosomes protein; tox., toxin; HNH, HNH family nuclease.
Fig. 6
Fig. 6
Diversity of domain organizations in the PrimPol-PV1 supergroup. The number of representative sequences having specific domain organization is indicated on the left. The catalytic helicase domains are shown in blue; primases, red; other domains, green. WH, winged HTH domain; αHD, α-helical domain; MCM, minichromosome maintenance helicase; SF2, superfamily 2 helicase.
Fig. 7
Fig. 7
Superimposition of pRN1 Primpol PriCT-1 domain (PDB: 3m1m), PriCT-2 domain of B. badius protein (WP_063441057), PriX of Sulfolobus solfataricus (PDB: 4wyh) and the N-terminal part (PriL Fe-S_N) of Saccharomyces cerevisiae primase large subunit Fe-S domain (PDB: 3lgb). Atoms of Fe-S cluster are shown as spheres.

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