DNA Polymerase Diversity Reveals Multiple Incursions of Polintons During Nematode Evolution

Abstract

Polintons are double-stranded DNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family but encode a distinct protein-primed DNA polymerase B (pPolB) that is related to homologs from a different group of Polintons present outside of the Nematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting interphylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of an HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.

Keywords: DNA polymerase; Polinton/Maverick; dsDNA virus; horizontal gene transfer; nematode; transposon.

Fig. 1.

Two distinct DNA polymerase B families in Caenorhabditis polintons. a) A phylogenetic tree (left) was constructed by applying a ML-based method (IQ-TREE) to a multiple sequence alignment of pPolB proteins from Polintons of 19 nematode species. The genetic architectures of these elements are shown on the right. These Polintons were chosen for initial sequence alignment based on uninterrupted DNA polymerase and other Polinton protein coding regions. Bootstrap supporting values are shown on the top position of tree branches. Abbreviations for inferred polinton components are as follows: PRO for adenovirus-like maturation protease, puORF for Polinton-uncharacterized ORF, ATPase for a packaging ATPase, MCP and mCP for major and minor capsid proteins, INT for a retroviral-like-element integrase, and TIR for terminal inverted repeat. "Uncharacterized" are novel (potentially either spurious or functional) ORFs that have not been previously described in other Polintons. Labels in parentheses denote unique identifiers (UIs) for each Polinton (see supplementary table S1, Supplementary Material online). b) A dot plot and a background box plot show percent (%) amino acid identities between each Polinton protein found in 49 C. briggsae Polintons.

Fig. 2.

HNH and VSR endonuclease domains are found in terrestrial and marine nematode pPolB2s, respectively, but not in pPolB1. a) Aligned, nonaligned, and gap parts of pPolB1 and pPolB2 (middle panel) from sequence-free structural alignment are shown with pLDDT (predicted local distance difference test) score predicted at the single amino acid level by AlphaFold2. Overall aligned parts for both pPolB1 and pPolB2 showed high pLDDT score, suggesting confident prediction of the structures. b) The predicted structure of C. briggsae pPolB2 (green) with the HNH endonuclease domain (red). c to e) The predicted structure of pPolB2-specific HNH domain (c) and the crystal structure of P. alcaligenes PacI HNH endonuclease (d) (PDBid: 3M7K) (Shen et al. 2010) are superimposed e) (RMSD: 3.03, TM-score: 0.55) to show potentially conserved structural folds. f) Aligned and non-aligned parts of C. briggsae pPolB2 and T. latispiculum pPolB2 (middle panel) from sequence-free structural alignment are shown with pLDDT score. g) The predicted of T. latispiculum pPolB2 (cyan) with the VSR domain (yellow). h to j) The predicted structure of T. latispiculum pPolB2 VSR endonuclease domain (h) and the crystal structure of Escherichia coli VSR endonuclease (i) (PDBid: 1VSR) (Tsutakawa et al. 1999) are superimposed (j) (RMSD: 3.06, TM-score: 0.63) to show potentially conserved structural folds. Dark and light colors for both orange and blue (c to e and h to j) indicate aligned and nonaligned positions, respectively.

Fig. 3.

Interspecies copy number variations of nematode Polintons. Bar graphs (right panel) showing average copy numbers per genome of full-length pPolB1-class and pPolB2-class Polintons in selected Nematoda genomes where the species can be placed on a current phylogenetic tree based on recent analyses (Ahmed et al. 2022; Lee et al. 2023). Note that several species that have not yet been placed on the tree are not shown here; all information for species (placed and not) is shown in supplementary table S1, Supplementary Material online.

Fig. 4.

Two distantly related groups of pPolBs in C. briggsae Polintons. a to c) Scatter plots showing percent amino acid identity of 1 protein for each axis from C. briggsae Polinton species; mCP and INT a), puORF and ATPase b), and pPolB1/2 and ATPase c). Each dot indicates a pair of 2 C. briggsae Polintons, whose proteins indicated at the X and Y axes were aligned, respectively, for calculating percent identity. Dot colors indicate whether 2 Polintons in a pair for comparison are within the same pPolB group or from different pPolB groups. d) Heatmap showing percentage amino acid identity of proteins. Each row shows an individual C. briggsae Polinton, while each column represents one of the conserved Polinton proteins. All comparisons were performed against a query C. briggsae Polinton (Plnt_Cbri_18). e) Genetic architectures of 4 C. briggsae Polintons (QX1410 strain, a wild isolate) shown with % amino acid identities for each Polinton protein. Plnt_Cbri_41 and Plnt_Cbri_43 are relatively close to each other, showing >90% identity, while Plnt_Cbri_43 and Plnt_Cbri_38 are more distant (evidenced by lower % identities). The continuous nature of the homology outside of the pPolB region provides evidence for gradual divergence in these regions during the ancestry of the current elements. By contrast, the Plnt_Cbri_35 Polinton showed strong similarity in all proteins except pPolB (particularly to PInt_Cbri_41), with dramatic divergence for pPolB, suggesting a swapping event of the pPolB gene during the derivation of this Polinton. f) A tanglegram showing the relationship between 2 phylogenetic trees constructed from multiple sequence alignment of C. briggsae pPolB1/2 (left panel) and Fusogen (right panel) proteins. The colors of the nodes indicate pPolB1-class and pPolB2-class. Dashed lines between 2 trees denote the correspondence of pPolB and Fusogen proteins found from the same Polinton. The tree exemplifies that close relationships between non-pPolB proteins can be accompanied by very distant relationships between the corresponding pPolBs.

Fig. 5.

Distinct distribution and evolutionary trajectories of pPolB1 and pPolB2 groups in the biosphere. a) A NCBI taxonomy tree with Eukaryotic taxa. The numbers in parentheses indicate the number of genomes having at least 1 copy of pPolB and the number of total genomes subjected to the Polinton pPolB search (see Materials and Methods for details). The sizes and colors in the pie charts indicate the copy number of Polinton pPolBs per pPolB-containing genomes and the classes of pPolB. b) A phylogenetic tree was constructed from multiple sequence alignment of pPolB proteins found in diverse taxa including viruses of prokaryotes, eukaryotes, DNA plasmids, and Polintons using the IQ-TREE-inferred ML method. Bootstrap supporting values are shown on the bottom positions of tree branches. All pPolBs identified in this study were found within Group 2 Polinton pPolBs where pPolB1 and pPolB2 classes form two major monophyletic groups. Asterisks indicated the Nematoda pPolB1 and pPolB2 proteins identified in this study.

Fig. 6.

Phylogenetic relationships of Polinton pPolB2 proteins from 3 distinct phyla. A phylogenetic tree (left) has been built by using IQ-TREE-inferred ML on a multiple sequence alignment of pPolB2 protein sequences found from Nematoda, Oomycota, and Mollusca (see supplementary fig. S9, Supplementary Material online for full alignment data). Bootstrap supporting values are shown on the top position of tree branches.