Jumping DNA polymerases in bacteriophages 

Jumping DNA polymerases in bacteriophages | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Jumping DNA polymerases in bacteriophages Natalya Yutin, Igor Tolstoy, Pascal Mutz, Yuri I Wolf, Mart Krupovic, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4452861/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Aug, 2024 Read the published version in Virology Journal → Version 1 posted 11 You are reading this latest preprint version Abstract Background Viruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families. Methods Groups of phages in which DNAP swapping likely occurred were identified as subtrees of a defined depth in a comprehensive evolutionary tree of tailed bacteriophages that included phages with DNAPs of different families. The DNAP swaps were validated by constrained tree analysis that was performed on phylogenetic tree of large terminase subunits, and the phage genomes encoding swapped DNAPs were aligned using Mauve. The structures of the discovered unusual DNAPs were predicted using AlphaFold2. Results We identified four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs “in situ”, without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage replication. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication. Conclusions Replacement of the DNAP gene by one encoding a DNAP of a different family occurred on many independent occasions during the evolution of different families of tailed phages, in some cases, resulting in very closely related phages encoding unrelated DNAPs. DNAP swapping was likely driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility. Evolution of Viruses DNA polymerases Bacterial antivirus defense Horizontal gene transfer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Viruses with large double-stranded (ds) DNA genomes in the realm Duplodnaviria share a uniformly conserved structural gene module but vary greatly in their repertoires of DNA replication proteins [ 1 , 2 ]. Some viruses encode most of the proteins required for DNA replication, whereas others rely (almost) entirely on the replication machinery of the host. Generally, the self-sufficiency of DNA replication correlates with the viral genome size. DNA polymerases (DNAPs) are central components of the viral replication systems that are present in more than half of the available genomes of duplodnaviruses greater than 40kb in size [ 3 ]. There are four major DNAP families involved in the genome replication in cellular life forms, families A, B, C and D (hereafter PolA-D), with the A, B and C families also being common among DNA viruses. The core catalytic domains of these DNAPs adopt three unrelated folds, namely, (i) the RNA Recognition Motif (RRM), often called the Palm domain (joins the accessory Thumb and Fingers domains) in PolA and PolB, (ii) nucleotidyltransferase Polβ-like fold in PolC, and (iii) the double-psi beta-barrel domain in PolD [ 4 – 8 ]. In bacteria, PolC is the primary polymerase responsible for the genome replication, whereas PolA is involved in DNA repair processes; PolB is rare in bacteria and is apparently derived from viruses [ 7 , 9 ]. In archaea, replication is catalyzed by either PolB or PolD, and paralogs of PolB are also involved in repair [ 10 ]. In eukaryotes, almost all processes of DNA synthesis involved in both replication and repair are catalyzed by DNAPs of the PolB family in the nucleus and PolA in mitochondria [ 11 , 12 ]. Different groups of tailed viruses of the class Caudoviricetes infecting bacteria and archaea encode PolA, PolB or PolC (or no DNAP at all), PolA being the most common, and PolC the rarest [ 3 ]. All large dsDNA viruses of eukaryotes, in the realms Duplodnaviria (phylum Peploviricota ) and Varidnaviria (phylum Nucleocytoviricota ), and unassigned class Naldaviricetes (baculo-like viruses), employ PolB [ 7 ]. Many smaller viruses (with < 50 kb genomes), especially in the realm Varidnaviria (e.g., polintons, adenoviruses, tectiviruses), replicate with the help of a distinct variety of B family DNAPs, the protein-primed PolB [ 3 , 13 – 15 ], or, less commonly, PolA, also referred to as TV-Pol [ 16 ]. Notably, archaeal viruses exclusively encode family B DNAPs which can be either RNA- or protein-primed [ 17 , 18 ]. The DNAPs are essential proteins that are highly conserved within each family, at the sequence and structure levels [ 3 , 6 ]. Therefore, it came as a surprise that among closely related genomes of phages in the order Crassvirales , multiple replacements of PolA with PolB and vice versa were observed [ 19 ]. Similar replacement of replication proteins was detected also among smaller phages of the order Vinavirales [ 20 , 21 ]. It is particularly notable that in each of these cases, the replacements occurred within otherwise conserved genomic contexts. In this work, we aimed to systematically identify and explore cases of between-family DNAP swapping in Caudoviricetes . We show that DNAPs were swapped repeatedly in the evolution of multiple groups of tailed phages. Methods Dataset of phage genomes and phage genome tree Genome-wide relationships between the 18,382 Caudoviricetes genomes, available in GenBank as of November 2022, were analyzed using reciprocal best hits between viral protein sequences as follows. The set of non-nested ORFs of at least 75 bp was obtained for each virus genome using the NCBI ORFfinder tool ( https://www.ncbi.nlm.nih.gov/orffinder/ ). Reciprocal best hits for all pairs of genomes ( A , B ), covering at least 50% of the query sequences were identified between the two ORF complements using BLASTP [ 22 ]. Distance between the two genomes was calculated as $${D}_{A,B}={D}_{B,A}=1-({C}_{A,B}+{C}_{B,A})/({L}_{A}+{L}_{B})$$ where C A,B is the total length of the part of genome A , covered by ORFs that have reciprocal best hits in genome B and L A , is the length of genome A (ditto for C B,A and L B ). The tree was reconstructed from the pairwise distance matrix using the FastMe 2.0 program [ 23 ] and ultrameterized by iteratively balancing subtrees, descending from each internal node. Identification of phage DNA polymerases PolA, PolB, PolC reference protein sequences were collected from the NCBI virus database ( https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/ ) and from the respective publications, in particular, the PolA sequences were from [ 15 , 24 , 25 ]; PolB sequences were from [ 7 ], and PolC sequences were from [ 26 ] ( https://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/ ). Open reading frames (ORFs) from the 18,382 Caudoviricetes genomes were searched for polymerases using BLASTP with collected reference PolA, PolB, PolC proteins as queries (e-value threshold of 0.0001). The initial set of hits was clustered using MMSEQS2 [ 27 ] at similarity threshold 0.5; sequences within clusters were aligned using MUSCLE5 [ 28 ]. Cluster alignments were iteratively compared to each other using HHSEARCH and aligned using HHALIGN [ 29 ]. Sequences in the final alignments were examined for the presence of the critical catalytic residues. Full-length sequences predicted to be catalytically active were retained for the downstream analysis (see Supplementary Table S1 for the final set of polymerases, classified as active). DNAP swapping hotspots were identified by the presence of DNAPs from different families within a subtree of depth 0.15 (corresponding to ~ 1/3rd of the total tree depth). Sister subtrees exhibiting polymerase diversity were grouped into DNAP-swapping clades. Comparison of phage genomes Pairwise genome alignments were constructed using Mauve [ 30 ] and visualized using with Geneious Prime® 2022.1.1 ( https://www.geneious.com ). Predicted phage proteins were annotated using CDD [ 31 ] and HHPRED [ 29 , 32 ]. Phylogenetic analysis of phage proteins The identified viral PolA, PolB, PolC sequences were combined with homologs identified in a collection of completely sequenced bacterial and archaeal genomes downloaded from NCBI Genomes ( https://ftp.ncbi.nlm.nih.gov/genomes/ASSEMBLY_REPORTS/ ) in November 2021. Sequences of phage DNAPs of the order Crassvirales were added from [ 19 ]. The protein sequences were aligned using MUSCLE5 [ 28 ]. Phylogenetic trees were constructed using IQ-TREE 2 [ 33 ], with the following models chosen according to BIC by the built-in model finder: VT + F + R10 for PolA, Q.pfam + F + R8 for PolB, and VT + F + R5 for PolC, and visualized with MEGA11 [ 34 ]. Large terminase subunits were aligned using MUSCLE5 [ 28 ]; constrained and unconstrained phylogenetic trees were reconstructed using IQ-TREE 2 [ 33 ] with the automatically selected evolutionary models and compared using the built-in Approximately Unbiased test. Protein structure prediction and analysis MSAs for divergent family A DNA polymerases identified in this study (divPolA1 and divPolA2) were submitted to a local installation of ColabFold (colabfold_batch with default settings except “–num-models 1 –num-recycle 3”) [ 35 ]. In addition, all individual divPolA1 and divPolA2 were modeled with a singularity version of AlphaFold2 [ 36 ] (version 2.2.0 with the following specifications: “--db_preset = full_dbs –model_preset = monomer_ptm –max_template_date = 2022-10-01”) on the high performance cluster BIOWULF at the NIH. All models were compared to a local version of pdb70 (created on December 10, 2021) using Dali [ 37 ] to identify closest structures. Structure-guided alignments between representative divPolAs and closest related structures were obtained using the Dali web server [ 38 ], and key residues were identified. Representative structures modeled with AlphaFold2 (divPolA1 clade5: CAB4155247, clade6: CAB4155247 and divPolA2 AUR84708) were displayed and superimposed with the respective DNAP structures from pdb using ChimeraX [ 39 ]. Results DNA polymerase diversity in Caudoviricetes In the analyzed set of 18,382 Caudoviricetes genomes, we identified 6560 PolA, 2857 PolB, and 947 PolC proteins (Supplementary Table S1 ). The Caudoviricetes genome tree was split into subtrees at the depth of 0.15, roughly corresponding to a genus level (see an example in Supplementary Figure S1 ; https://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/genome_tree/ ). Of the 1,514 subtrees that included more than one virus genome, 563 were found to encode a DNAP, and 8 encoded more than one DNAP. Analysis of the DNAP distribution pattern revealed two distinct types of DNAP heterogeneities within phage subtrees: (i) DNAPs of different families were encoded in closely related viruses, with a single copy in each genome, implying swapping of DNAP genes (6 subtrees), and (ii) the same viral genome encoded two DNAPs of different families (2 subtrees). DNA polymerase swapping in Caudoviricetes We identified 6 subtrees in the phage genome tree in which the phages encoded DNAPs of different families. Representative pairs of most closely related genomes with different family DNAPs from these subtrees are listed in Table 1 . To investigate the provenance of these groups of phages encoding distinct DNAPs, we examined deeper clades in the phage genome tree that included each of the 6 subtrees (Table 1 ), apart from Crassvirales for which frequent PolA-PolB swaps have been reported previously [ 19 ]. Clades 1, 2, and 3 consisted of PolA- and PolB-encoding genomes, and clade 4 genomes encompassed PolA and PolC (Figs. 1 , 2 ). The DNAPs from these clades were reciprocally mapped onto the corresponding PolA, PolB, and PolC trees (Supplementary Figure S2 ). Examination of this mapping suggests that, in addition to inter-family DNAP swaps, some intra-family DNAP swaps occurred in the selected clades, that is, PolA or PolB was apparently replaced by a distinct DNAP of the same family on several occasions. Below we discuss each of these clades in detail, in an attempt to reconstruct the evolutionary scenarios. Table 1 Phage clades displaying DNAP swapping and examples of exchange between closely related genomes DNAP genome Organism genome length ANI or AAI clade tree00180 PolA MN184886.1 Erwinia phage pEp_SNUABM_08 62716 39% AAI Clade1; subtrees 00180–00189 PolB MN505213.1 Serratia phage JS26 63971 tree00183 PolA KF626665.1 Phage Sano 56147 39% AAI --“-- PolB MT161385.1 Xanthomonas phage FoX4 60418 tree00184 PolA MN536026.1 Pseudomonas phage vB_Pae-SS2019XI 57567 38% AAI --“-- PolB MF959998.1 Marinobacter phage PS6 58226 tree01348 PolA MZ477002.1 Acinetobacter phage Phab24 93604 92.72% ANI Clade2; subtrees 01336–01351 PolB MN276049.1 Acinetobacter phage BS46 94068 tree01419 PolA MG592602.1 Vibrio phage 1.237.B._10N.261.52.C5 60160 99.54% ANI Clade3; subtrees 01419–01425 PolB MG592464.1 Vibrio phage 1.089.O._10N.261.51.F9 59851 tree01472 PolA MT601273.1 Bacillus phage vB_BsuS-Goe12 124287 99.74% ANI Clade4; subtrees 01466–01481 PolC MT601274.1 Bacillus phage vB_BsuS-Goe13 126848 We sought to validate the intra-clade cross-family swaps of DNAPs using the large subunit of the terminase (TerL) as the reference. The TerL sequences were collected from the genomes with identified DNAPs within each of the clades 1, 2, 3 and 4 and clade-specific TerL phylogenetic trees were constructed. Then, we constructed topologically constrained trees, separating the TerL from genomes encoding different DNAPs (for example, for clade 1, the constraint separated TerL from PolA- and PolB-bearing genomes). Such constrained topologies represent hypothetical phylogenies where DNAPs of different families are not intermixed within the clade histories. The optimal TerL trees satisfying these constraints were compared to the unconstrained trees, in an attempt to falsify the scenario with multiple DNAP swaps. For all clades, the Approximately Unbiased test decisively rejected the constrained topologies (Supplementary Table S2 ), suggesting that multiple DNAP swaps within each clade did occur. Clade 1 included 126 genomes from several genera of the family Casjensviridae (genome size range 50–70 kb) of which 96 encoded PolA whereas the remaining 30 encoded PolB. The genome tree for this clade is dominated by a distinct group of PolA (A1 in Fig. 1 ) in which 3 disjointed PolB branches (B1-B3) and two branches from a separate group of PolA (A2) are embedded. Altogether, comparison of the phage genome tree with the phylogenetic trees of PolA and PolB suggests 8 independent DNAP swaps including both 6 inter-family (PolA to PolB) exchanges and 2 intra-family (A1 to A2) exchanges (Fig. 1 ). In most of the phage genomes in this clade, the swapped PolA and PolB genes share the same or similar genomic neighborhood (Fig. 1 ). Clade 2 (subtrees 01336–01351 in the phage genome tree) unites phages from genera Plaisancevirus , Saclayvirus , Barbavirus , subfamily Ounavirinae , and several unclassified Caudoviricetes (genome size range 80–105 kb). The phages in subtree 01347 encode an additional protein with remote sequence and structural similarity to PolA, which we discuss in the next section. Here we address the apparent PolA to PolB swaps in this clade (Fig. 2 ). PolBs of Clade 2 are monophyletic (B4 in Fig. 2 ), but PolAs come from three distinct branches (A3, A4 and A5 in Fig. 2 ; see Supplementary Figure S2 for the DNAP trees). As in Clade 1, comparison of the phage genome tree with PolA and PolB phylogenetic trees suggests several independent swaps although the direction and the order of these events is difficult to establish. Two Acinetobacter phages of Clade 2, Phab24 (MZ477002) and BS46 (MN276049), have average nucleotide identity (ANI) of 93%, and yet, encode DNAPs of different families, PolA and PolB, respectively (Table 1 ; Fig. 3 a). Comparison of the genome organization in the vicinity of the DNAP genes (in which we additionally included the corresponding genome region of Acinetobacter phage TaPaz (MZ043613) because its PolB is most similar to PolB of MN276049 (Acinetobacter phage BS46). suggests that, in this case, the primase-helicase gene was replaced along with the DNAP. In the upper part of each panel, nucleotide sequence similarity between the compared genomes is shown in green or yellow, on the scale from 0 to 100%. Red arrows denote the genomic regions containing the DNAP genes, visualized on the genome maps. The lower parts, show the blow up of the regions containing the DNAP genes. Functionally annotated homologous genes are shown in the same colors. DNAP genes are labeled with their GenBank protein IDs. Grey shading highlights genomic regions with high nucleotide sequence similarity (percent identity indicated). Pink lines connect genes with significant detectable amino acid sequence similarity detected with BLASTP but no significant nucleotide sequence similarity. Clade 3 unites subtrees 01419–01425 (Fig. 2 ) and includes phages from genera Sashavirus , Nonanavirus , Gorganvirus , and unclassified Caudoviricetes , with genome size range of 42.5–62.5 kb. In this clade, a single PolB to PolA swap appears to have occurred within the subtree01419, whereas the basal PolB genes belong to distinct clades (Fig. 2 ). In this clade, we identified a pair of nearly identical genomes, Vibrio phage 1.237.B._10N.261.52.C5 (MG592602) and Vibrio phage 1.089.O._10N.261.51.F9 (MG592464), that encode different family DNAPs, PolA and PolB, respectively. Pairwise genome comparison shows that the DNAP neighborhood is the only large region with markedly lower similarity between the two genomes (Fig. 3 b). The DNAP gene is the only one that obviously was replaced but additional rearrangements might have occurred in the adjacent genomic region containing genes encoding uncharacterized small proteins (Fig. 3 b). Clade 4 unites subtrees 01466–1481 including families Tybeckvirinae and Andrewesvirinae , genera Audreyjarvisvirus , Spbetavirus , Latrobevirus , Sextaecvirus , Slashvirus , and several unclassified Caudoviricetes (genome size range 61–185 kb). In this clade, the phages encode either PolC or PolA. PolC, specifically group C1, is likely to be ancestral in this assemblage of phages (Fig. 2 and Supplementary Figure S2 ). This ancestral PolC apparently was replaced with PolA on two independent occasions (Fig. 2 ), and furthermore, underwent several intra-family replacements involving PolC variants from groups C2 and C3. We also identified a pair of closely related genomes in this clade with over 99% ANI, Bacillus phage vB_BsuS-Goe12 (MT601273) and Bacillus phage vB_BsuS-Goe13 (MT601274), that encode DNAPs of different families, PolA and PolC, respectively. Comparative genome analysis of this pair of phages revealed an extended segment of dissimilarity suggesting that several replicative genes, including DnaB-like helicase, DnaG-like primase and RecJ-like exonuclease, traveled together with the DNAPs, but the replacement keeps the gene context unchanged, that is, the genes appear to have been replaced en bloc (Fig. 3 c). When the inter-family swaps were found to have occurred on shallow branches of the virus tree, these events typically involved phages that shared bacterial hosts (Supplementary Figure S3 ). Two novel phage PolAs In addition to the typical DNAPs, we identified two divergent variants of PolA encoded in phage genomes. One of these, denoted divPolA1, is present in a group of 14 phages (Flavobacterium phage vB_FspM_immuto_3-5A and related phages), with genomes in the range of 155–190 kb that also encode a ‘regular’ DNAP, either family A, B, or C (Fig. 4 a). The conserved arrangement of genes implicated in replication downstream of the divPolA1 gene suggests that divPolA1 is involved in replication (Fig. 4 b). Structural prediction for divPolA1 (Fig. 5 ) showed that it contains a Palm domain in which the main catalytic residues are conserved, whereas the Thumb domain is truncated and the 3’ exonuclease domain seems to be inactivated, with all three catalytic aspartates replaced (Fig. 5 ). Thus, divPolA1 most likely retains the DNAP activity whereas the exonuclease activity is lost. Another diverged PolA variant, divPolA2, was initially identified in 110 genomes of barbaviruses (Rheinheimera phage vB_RspM_Barba18A and related viruses) with genomes of 80–85 kb, each also encoding a regular PolB. Additional PSI-BLAST searches against the Caudoviricetes database using barbavirus divPolA2s as queries revealed more divPolA2 proteins, sometimes with two or three paralogs per phage genome (Fig. 6 ). Unlike the PolB of these phages, which is embedded within a typical context of replication-related genes, the gene encoding divPolA2 is located in variable gene neighborhood. Structural modeling suggests that divPolA2 contains an active DNAP (Palm) catalytic domain but lacks a Thumb domain homologous to those of any other DNAPs (Fig. 7 ). Instead, this protein contains an N-terminal globular domain without detectable similarity to any other known domains that potentially might function as the Thumb. As in the case of divPolA1, the 3’ exonuclease domain is lacking. Most likely, divPolA2 is not the replicative enzyme of barbaviruses, a role that belongs to PolB. Instead, divPolA2 might be a DNAP involved in repair processes, or an RNA polymerase, given that PolA was co-opted for that function in T7 and related phages [ 5 ]. Of note, structural comparison did not only reveal DNAPs as the top hits for divPolA2, but also DNA-directed RNA polymerases (mitochondrial RNA polymerase (PDB ids: 7a8p, 6ymv, Dali z-score ~ 12) and, with lower z-score (~ 9), also a viral DNA-directed RNA polymerase from bacteriophage N4 (genus Enquatrovirus , class Caudoviricetes ) (PDB id: 4ff3). These observations are compatible with the possibility that divPolA2 is actually an RNA polymerase although the N-terminal globular domain of divPolA2 is unrelated to the N-terminal domains of PolA-related RNA polymerases (Fig. 7 ). Discussion Tailed viruses of bacteria and archaea that comprise the class Caudoviricetes in the realm Duplodnaviria are considered to be the most abundant group of viruses on earth [ 40 , 41 ]. Although the virion structures and the core structural proteins are conserved throughout the realm, these viruses greatly differ in their genome size and gene repertoires. In particular, some caudoviricetes encode a (nearly) complete suite of proteins required for replication, whereas others have none, and the entire range of intermediates exists as well [ 3 ]. This variety notwithstanding, more than half of the caudoviricetes encode a DNAP – the obvious centerpiece of the replication machinery – that belongs to either A or B, or C family. Generally, the DNAP is a conserved component of the replication apparatus. Unexpectedly, however, in our previous comparative genomic analysis of Crassvirales (the order of Caudoviricetes that includes the most abundant viruses identified in the human gut), we found that DNAPs were swapped between closely related phages on multiple occasions, with PolB replacing PolA or vice versa [ 19 ]. Intrigued by this observation, we probed a much broader range of phages and report here that multiple DNAP swaps occurred in at least four additional phage groups. The DNAP replacements involved either different families, that is, PolA to PolB and vice versa, as well as PolC to PolA, or distinct groups within the same DNAP family. Remarkably, these replacements in each case occurred “in situ”, without a change in the neighboring gene arrangement. The swap involved either the DNAP gene alone or several adjacent genes encoding other components of the replication machinery, but in each case, the gene replacement appears to have occurred with “surgical precision”. The genes for proteins involved in replication tend to cluster in viral genomes [ 3 ], and the preservation of their order upon DNAP swapping implies that coregulation of these genes is important for phage reproduction. The recurrent DNAP swapping in phage evolution raises intriguing questions on both the molecular mechanisms of these exchanges and the selective forces that could drive them. The mechanisms of DNAP swapping remain enigmatic considering the striking precision of these events. Whether or not the phages involved in the swaps are within the range of sequence identity required for homologous recombination, it hardly can contribute to the capture of distantly related genes. Whether the replacing DNAP comes from a prophage integrated in the host cell genome or a coinfecting phage, illegitimate recombination seems to be essential, and the positive selection associated with the swap should be strong enough to provide for the fixation of the rarely emerging precise replacements. In cases where we could pinpoint the intra-family DNAP swaps to a narrow phylogenetic context (that is, between closely related phages), they typically occurred between phages that infect the same host (at least up to the genus level; Supplementary Figure S3 ). These observations are compatible with the involvement of coinfection, either cotemporaneous or sequential, in DNAP exchanges. With respect to the evolutionary forces driving DNAP swapping, it has been shown that multiple defense systems specifically target the phage replication machinery components [ 42 ]. Involvement of at least four types of known defense mechanisms can be suspected. Mutations within DNAP genes have been demonstrated to allow phages to escape restriction by the poorly understood Borvo defense system [ 42 , 43 ]. Although the mechanism of Borvo activation remains unclear, it has been suggested that the DNAP structure, its complex with other proteins and/or DNA encompasses molecular patterns that activate Borvo [ 44 ]. Similarly, AbiQ, a type III toxin-antitoxin abortive infection system, was shown to be activated by various phage proteins, with escape mutants localized to a family A DNAP [ 45 ]. Another recent study has similarly shown that mutations in PolB of T-even phages enabled escape from DarTG, a type II toxin-antitoxin system that provides immunity by ADP-ribosylating phage DNA [ 46 ]. Furthermore, pattern recognition systems, in particular, those centered at antivirus STAND ATPases (Avs), have been shown to target conserved viral structural proteins, such as the terminase large subunit and the portal protein [ 47 , 48 ]. These viral proteins are conserved at the level of structures even if their sequences diverge relatively fast. The DNAPs, although not universal among tailed phages, unlike terminase and portal, are common and even more highly conserved at the sequence level, and therefore, the existence of multiple pattern recognition systems targeting DNAPs appears likely. The high sequence conservation of the DNAPs within each family suggests potential involvement of another type of defense, namely, adaptive immunity mediated by CRISPR systems, and more specifically, primed adaptation [ 49 , 50 ]. CRISPR spacers targeting conserved sequences in the DNAP genes are likely to retain complementarity level sufficient for primed adaptation longer than they do in the case of less conserved genes, facilitating acquisition of immunity to the respective phages. Furthermore, existence of yet unknown defense mechanisms targeting DNAPs remains a possibility. An additional or alternative driver of replication module swapping between phages could be the incompatibility of closely related replicons within a coinfected cell, analogous to plasmid incompatibility [ 51 , 52 ]. Further study of the notable but not yet well understood phenomenon of DNAP swapping in phages has the potential to reveal unknown facets of interactions between phages and their bacterial hosts as well as conflicts among different phages. Conclusions We show in this work that replacement of DNAPs by distantly related or even unrelated ones is common in the evolution of tailed phages of the class Caudovirecetes . Remarkably, DNAP swapping always occurs “in situ”, with the organization of the surrounding genes, typically, encoding other proteins involved in phage genome replication being preserved, whether the DNAP gene is the only region of substantial divergence between closely related phage genomes, or the replacement involves several neighboring genes. We hypothesize that although illegitimate recombination is required for replacement of the DNAP genes, selection driving such replacements is strong enough to allow the rare emerging variants with precise insertion of the new sequence to be fixed in the phage population. The factors underlying this selection likely include avoidance of host defense mechanism, such as Borvo, pattern recognition or CRISPR primed adaptation, that target DNAPs. In addition to DNAP swapping, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials This work is based on the analysis of genomes publicly available in GenBank. All other data generated by this analysis are contained in the Supplementary Material or publicly available at https://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/ Competing interests The authors declare no competing interests. Funding N.Y., P.M., I.T., Y.I.W. and E.V.K. are funded by the Intramural Research Program of the National Institutes of Health (National Library of Medicine). Authors’ contributions N.Y. and E.V.K. conceptualized the project; N.Y., I.T., and Y.I.W. developed the methodology; N.Y., P.M., Y. I. W., M.K. and E.V.K. analyzed the data; N.Y. and E.V.K. wrote the manuscript; all authors edited and approved the manuscript. Acknowledgements This work utilized the computational resources of the NIH HPC Biowulf cluster ( http://hpc.nih.gov ). References Koonin EV, Dolja VV, Krupovic M, Varsani A, Wolf YI, Yutin N, Zerbini FM, Kuhn JH: Global Organization and Proposed Megataxonomy of the Virus World . Microbiol Mol Biol Rev 2020, 84 (2)::e00061-00019. Weigel C, Seitz H: Bacteriophage replication modules . FEMS Microbiol Rev 2006, 30 (3):321-381. Kazlauskas D, Krupovic M, Venclovas C: The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes . Nucleic Acids Res 2016, 44 (10):4551-4564. Koonin EV, Krupovic M, Ishino S, Ishino Y: The replication machinery of LUCA: Common origin of DNA replication and transcription BMC Biology 2020, in press . Czernecki D, Nourisson A, Legrand P, Delarue M: Reclassification of family A DNA polymerases reveals novel functional subfamilies and distinctive structural features . Nucleic Acids Res 2023, 51 (9):4488-4507. Raia P, Delarue M, Sauguet L: An updated structural classification of replicative DNA polymerases . Biochem Soc Trans 2019, 47 (1):239-249. Kazlauskas D, Krupovic M, Guglielmini J, Forterre P, Venclovas C: Diversity and evolution of B-family DNA polymerases . Nucleic Acids Res 2020, 48 (18):10142-10156. Sauguet L: The Extended "Two-Barrel" Polymerases Superfamily: Structure, Function and Evolution . J Mol Biol 2019, 431 (20):4167-4183. Kornberg A, Baker TS: DNA Replication , 2nd edn. San Francisco: Freeman; 1992. Greci MD, Bell SD: Archaeal DNA Replication . Annu Rev Microbiol 2020, 74 :65-80. Burgers PM: Polymerase dynamics at the eukaryotic DNA replication fork . J Biol Chem 2009, 284 (7):4041-4045. Burgers PMJ, Kunkel TA: Eukaryotic DNA Replication Fork . Annu Rev Biochem 2017, 86 :417-438. Krupovic M, Koonin EV: Polintons: a hotbed of eukaryotic virus, transposon and plasmid evolution . Nat Rev Microbiol 2015, 13 (2):105-115. Schoenfeld TW, Murugapiran SK, Dodsworth JA, Floyd S, Lodes M, Mead DA, Hedlund BP: Lateral gene transfer of family A DNA polymerases between thermophilic viruses, aquificae, and apicomplexa . Mol Biol Evol 2013, 30 (7):1653-1664. Nasko DJ, Chopyk J, Sakowski EG, Ferrell BD, Polson SW, Wommack KE: Family A DNA Polymerase Phylogeny Uncovers Diversity and Replication Gene Organization in the Virioplankton . Front Microbiol 2018, 9 :3053. Iyer LM, Abhiman S, Aravind L: A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases . Biol Direct 2008, 3 :39. Makarova KS, Krupovic M, Koonin EV: Evolution of replicative DNA polymerases in archaea and their contributions to the eukaryotic replication machinery . Frontiers Microbiol 2014, doi: 10.3389/fmicb.2014.00354 . Prangishvili D, Bamford DH, Forterre P, Iranzo J, Koonin EV, Krupovic M: The enigmatic archaeal virosphere . Nat Rev Microbiol 2017, 15 (12):724-739. Yutin N, Benler S, Shmakov SA, Wolf YI, Tolstoy I, Rayko M, Antipov D, Pevzner PA, KooninE.V.: Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic features Nature Communications 2021, 12 :1044 Krupovic M, Bamford DH: Putative prophages related to lytic tailless marine dsDNA phage PM2 are widespread in the genomes of aquatic bacteria . BMC Genomics 2007, 8 :236. Yutin N, Rayko M, Antipov D, Mutz P, Wolf YI, Krupovic M, Koonin EV: Varidnaviruses in the Human Gut: A Major Expansion of the Order Vinavirales . Viruses 2022, 14 (9). Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs . Nucleic Acids Res 1997, 25 (17):3389-3402. Lefort V, Desper R, Gascuel O: FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program . Mol Biol Evol 2015, 32 (10):2798-2800. Keown RA, Dums JT, Brumm PJ, MacDonald J, Mead DA, Ferrell BD, Moore RM, Harrison AO, Polson SW, Wommack KE: Novel Viral DNA Polymerases From Metagenomes Suggest Genomic Sources of Strand-Displacing Biochemical Phenotypes . Front Microbiol 2022, 13 :858366. Dorawa S, Werbowy O, Plotka M, Kaczorowska AK, Makowska J, Kozlowski LP, Fridjonsson OH, Hreggvidsson GO, Aevarsson A, Kaczorowski T: Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity . Int J Mol Sci 2022, 23 (14). Timinskas K, Venclovas C: New insights into the structures and interactions of bacterial Y-family DNA polymerases . Nucleic Acids Res 2019, 47 (9):4393-4405. Steinegger M, Soding J: MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets . Nat Biotechnol 2017, 35 (11):1026-1028. Edgar RC: Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny . Nat Commun 2022, 13 (1):6968. Zimmermann L, Stephens A, Nam SZ, Rau D, Kubler J, Lozajic M, Gabler F, Soding J, Lupas AN, Alva V: A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core . J Mol Biol 2018, 430 (15):2237-2243. Darling AC, Mau B, Blattner FR, Perna NT: Mauve: multiple alignment of conserved genomic sequence with rearrangements . Genome Res 2004, 14 (7):1394-1403. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR et al : CDD/SPARCLE: functional classification of proteins via subfamily domain architectures . Nucleic Acids Res 2017, 45 (D1):D200-D203. Soding J: Protein homology detection by HMM-HMM comparison . Bioinformatics 2005, 21 (7):951-960. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, Lanfear R: IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era . Mol Biol Evol 2020, 37 (5):1530-1534. Tamura K, Stecher G, Kumar S: MEGA11: Molecular Evolutionary Genetics Analysis Version 11 . Mol Biol Evol 2021, 38 (7):3022-3027. Mirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M: ColabFold: making protein folding accessible to all . Nat Methods 2022, 19 (6):679-682. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A et al : Highly accurate protein structure prediction with AlphaFold . Nature 2021, 596 (7873):583-589. Holm L: DALI and the persistence of protein shape . Protein Sci 2020, 29 (1):128-140. Holm L: Dali server: structural unification of protein families . Nucleic Acids Res 2022, 50 (W1):W210-W215. Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE: UCSF ChimeraX: Structure visualization for researchers, educators, and developers . Protein Sci 2021, 30 (1):70-82. Suttle CA: Viruses in the sea . Nature 2005, 437 (7057):356-361. Mushegian AR: Are There 10(31) Virus Particles on Earth, or More, or Fewer? J Bacteriol 2020, 202 (9). Stokar-Avihail A, Fedorenko T, Hor J, Garb J, Leavitt A, Millman A, Shulman G, Wojtania N, Melamed S, Amitai G et al : Discovery of phage determinants that confer sensitivity to bacterial immune systems . Cell 2023, 186 (9):1863-1876 e1816. Millman A, Melamed S, Leavitt A, Doron S, Bernheim A, Hor J, Garb J, Bechon N, Brandis A, Lopatina A et al : An expanded arsenal of immune systems that protect bacteria from phages . Cell Host Microbe 2022, 30 (11):1556-1569 e1555. Huiting E, Bondy-Denomy J: Defining the expanding mechanisms of phage-mediated activation of bacterial immunity . Curr Opin Microbiol 2023, 74 :102325. Samson JE, Belanger M, Moineau S: Effect of the abortive infection mechanism and type III toxin/antitoxin system AbiQ on the lytic cycle of Lactococcus lactis phages . J Bacteriol 2013, 195 (17):3947-3956. LeRoux M, Srikant S, Teodoro GIC, Zhang T, Littlehale ML, Doron S, Badiee M, Leung AKL, Sorek R, Laub MT: The DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA . Nat Microbiol 2022, 7 (7):1028-1040. Gao LA, Wilkinson ME, Strecker J, Makarova KS, Macrae RK, Koonin EV, Zhang F: Prokaryotic innate immunity through pattern recognition of conserved viral proteins . Science 2022, 377 (6607):eabm4096. Kibby EM, Conte AN, Burroughs AM, Nagy TA, Vargas JA, Whalen LA, Aravind L, Whiteley AT: Bacterial NLR-related proteins protect against phage . Cell 2023, 186 (11):2410-2424 e2418. Jackson SA, Birkholz N, Malone LM, Fineran PC: Imprecise Spacer Acquisition Generates CRISPR-Cas Immune Diversity through Primed Adaptation . Cell Host Microbe 2019, 25 (2):250-260 e254. Shiriaeva AA, Kuznedelov K, Fedorov I, Musharova O, Khvostikov T, Tsoy Y, Kurilovich E, Smith GR, Semenova E, Severinov K: Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system . Sci Adv 2022, 8 (47):eabn8650. Igler C, Huisman JS, Siedentop B, Bonhoeffer S, Lehtinen S: Plasmid co-infection: linking biological mechanisms to ecological and evolutionary dynamics . Philos Trans R Soc Lond B Biol Sci 2022, 377 (1842):20200478. Pilosof S: Conceptualizing microbe-plasmid communities as complex adaptive systems . Trends Microbiol 2023, 31 (7):672-680. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1.xlsx SupplementaryTableS2.xlsx yutinDNAPs2024supplfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 26 Aug, 2024 Read the published version in Virology Journal → Version 1 posted Editorial decision: Revision requested 08 Jul, 2024 Reviews received at journal 06 Jul, 2024 Reviews received at journal 03 Jul, 2024 Reviewers agreed at journal 26 Jun, 2024 Reviewers agreed at journal 25 Jun, 2024 Reviews received at journal 03 Jun, 2024 Reviewers agreed at journal 27 May, 2024 Reviewers invited by journal 27 May, 2024 Editor assigned by journal 26 May, 2024 Submission checks completed at journal 26 May, 2024 First submitted to journal 21 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4452861","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310189169,"identity":"7428bda9-cb77-438d-84a0-02f4c9d087e0","order_by":0,"name":"Natalya Yutin","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Natalya","middleName":"","lastName":"Yutin","suffix":""},{"id":310189170,"identity":"4b210819-db87-4dcd-93c7-0b6b00e9fc0e","order_by":1,"name":"Igor Tolstoy","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Igor","middleName":"","lastName":"Tolstoy","suffix":""},{"id":310189171,"identity":"e118bfc1-9a64-4d5b-a5ed-3168100ecef6","order_by":2,"name":"Pascal Mutz","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pascal","middleName":"","lastName":"Mutz","suffix":""},{"id":310189172,"identity":"8e49e96a-701b-40be-9748-975b1843c9bf","order_by":3,"name":"Yuri I Wolf","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuri","middleName":"I","lastName":"Wolf","suffix":""},{"id":310189173,"identity":"24efc6d9-6234-44e6-989c-2d6be0e8cc0c","order_by":4,"name":"Mart Krupovic","email":"","orcid":"","institution":"Institut Pasteur","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mart","middleName":"","lastName":"Krupovic","suffix":""},{"id":310189174,"identity":"1c80a3e1-1b61-4026-86fd-1f2dcd0c52a6","order_by":5,"name":"Eugene V Koonin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACCRCRwGADZDE2MDAUAHnMPERpSYNqMSBWCwPDYSgLpIWBgBbJ9uZjDx7uOJ/HP7u5gbnAYBuDOTvvAcYvFYdxapHmOZZukHjmdrHEnYMNzDMMbjNYNvMlMMucwa1FTiLHTCKx7XbiBonEBmYeoBaDwzwGzJJtaYS0nCNBizREywFULYwf22xwe7/nWBpQS3LiDKBfgIpv84C0HGY4g1uLxPHmY5I/2+wS+2e3P3zMU3FbzuD8GcOHPyokcGpBAQcYoJFymEDUYAGMP0jWMgpGwSgYBcMYAAABHk9fgmpqBgAAAABJRU5ErkJggg==","orcid":"","institution":"National Institutes of Health","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Eugene","middleName":"V","lastName":"Koonin","suffix":""}],"badges":[],"createdAt":"2024-05-21 07:21:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4452861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4452861/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12985-024-02482-z","type":"published","date":"2024-08-26T15:57:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58145481,"identity":"0b072846-6de8-487d-b044-3a7ab68383a2","added_by":"auto","created_at":"2024-06-11 18:33:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147243,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA polymerase swapping in Clade 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLeft\u003c/em\u003e: Clade 1 genome tree, reduced to salient representatives. DNAP families and clades are marked on tree leaves; tree edge colors indicate the polymerase families; inferred DNAP swapping events are marked on the corresponding tree edges. \u003cem\u003eRight\u003c/em\u003e: genome maps of polymerase neighborhoods; homologous genes are shown in the same colors.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/b8a290b73a68fa5009e0f6e4.png"},{"id":58145480,"identity":"e1737f84-6fa2-4edb-9e1f-cd4408d25885","added_by":"auto","created_at":"2024-06-11 18:33:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA polymerase swapping in Clades 2, 3 and 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome trees of Clades 2, 3 and 4, reduced to salient representatives. DNAP families and clades are marked on tree leaves; tree edge colors indicate the polymerase families; inferred DNAP swapping events are marked on the corresponding tree edges.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/55db8075286900cc5aaffe85.png"},{"id":58147082,"identity":"4c675649-47c7-4eed-b815-b09afe37e191","added_by":"auto","created_at":"2024-06-11 18:41:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePairwise alignments of closely related viral genomes encoding DNAPs of different families\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Clade 2, tree01348 (unclassified Caudoviricetes). MN276049 is permuted at 27000; MZ477002 is reversed.\u003c/p\u003e\n\u003cp\u003eB. Clade 3, tree01419 (unclassified Caudoviricetes).\u003c/p\u003e\n\u003cp\u003eC. Clade 4, tree01472 (Spbetavirus).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/bdef831092b97d470362f4c7.png"},{"id":58145486,"identity":"4b039e83-adf1-490e-bc56-d048e6aa0608","added_by":"auto","created_at":"2024-06-11 18:33:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57012,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic and genomic context of divPolA1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, Genome tree of divPolA1-encoding viruses. Tree branches are marked according to the identity of the ‘regular’ polymerases: red, PolA; blue, PolB; green, PolC.\u003c/p\u003e\n\u003cp\u003eB, divPolA1 genome neighborhoods; homologous genes are shown in the same colors. Genomes: LR796625, LR796859, LR797270, LR796697, LR796420, LR796421, LR796188, LR796345: uncultured Caudovirales phages; MW353177: Flavobacterium phage vB_FspM_immuto_13-6C; MW353176: Flavobacterium phage vB_FspM_immuto_3-5A; MW353175: Flavobacterium phage vB_FspM_immuto_2-6A; MK892766: Prokaryotic dsDNA virus sp. isolate GOV_bin_1807; MK892784: Prokaryotic dsDNA virus sp. isolate GOV_bin_703; MK892584: Prokaryotic dsDNA virus sp. isolate GOV_bin_630.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/0a83661bf539dc413c49527b.png"},{"id":58147083,"identity":"56894c28-3206-4605-acde-8643f4759082","added_by":"auto","created_at":"2024-06-11 18:41:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":205049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edivPolA1 structure prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA, D. Predicted representative divPolA1 structures colored according to plddt score (AlphaFold2 model; A: CAB4155247, genome ID: LR796625; D: QDP51333, genome ID: MK892584).\u003c/p\u003e\n\u003cp\u003eB, E. Structural comparison of divPolA1 (blue) and a representative DNA polymerase I (pdb 1d9f, Klenow fragment). The representative DNAP I is colored by domain organization: 3’ exonuclease domain (3’ Exo, dark green), thumb domain (cyan) and palm/finger domain (green). Sites of motifs A, B and C highlighted in magenta/grey, orange/black and purple/light grey for DNAP I and divPolA1, respectively. Aspartic acid residues in 3’ exonuclease motifs I, II and III of DNAP I are highlighted in red, the corresponding sites in divPolA1 in black.\u003c/p\u003e\n\u003cp\u003eC, F. Structure-guided alignments of selected 3’ exonuclease and palm/finger domain motifs between divPolA1 representative (C: CAB4155247, genome ID: LR796625; F: QDP51333, genome ID: MK892584) and PolA 1D9F_A. Key residues highlighted in red.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/2b28a55230caf6a819b39a81.png"},{"id":58145488,"identity":"d856fb26-a87f-4be0-a409-1331b4c056d0","added_by":"auto","created_at":"2024-06-11 18:33:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":204101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic context of divPolA2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenome tree for the clade containing divPolA2 genes is shown. Arcs indicate subtrees. Numbers at tree tips indicate the number of divPolA2 paralogs. Barbaviruses are located in the subtree 01347.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/9b78f2de963dfb0c41875444.png"},{"id":58145483,"identity":"d4f872ca-c7f0-406c-b6c1-8b6daf3c22b7","added_by":"auto","created_at":"2024-06-11 18:33:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":889879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edivPolA2 structure prediction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Predicted representative divPolA2 structure colored according to plddt score (AlphaFold2 model; AUR84708, genome MG592441.1).\u003c/p\u003e\n\u003cp\u003eB,C. Structural comparison of divPolA2 (blue) and a representative DNA polymerase I (pdb 4b9v, B) and a representative RNA polymerase (pdb 7a8p, human mitochondrial RNAP, C). The representative DNAP I and RNAP are colored by domain organization: 3’ exonuclease domain (3’ Exo, dark green, DNAP I only), thumb domain (cyan) and palm/finger domain (green) and RNAP N-terminal pentatricopeptide domain (PPR, cornflower blue). Sites of motifs A, B and C highlighted in magenta/grey, orange/black and purple/light grey for divPolA2 and DNAP I, respectively.\u003c/p\u003e\n\u003cp\u003eD. Structure-guided alignments of selected motifs of palm/finger domain between divPolA2 and DNAP I 4b9v and RNAP (7a8p). Key residues highlighted in red.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/d56fc030f82b940883c8ede0.png"},{"id":63821071,"identity":"3614116f-86e9-4aab-8a89-07e041055e1e","added_by":"auto","created_at":"2024-09-02 16:11:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3498721,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/91deb6ab-6af1-4f10-a551-5999f5ed6682.pdf"},{"id":58145482,"identity":"0c06e19c-9ba6-45ad-a4eb-3d76b81d1067","added_by":"auto","created_at":"2024-06-11 18:33:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3597855,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/e968a2a0d05f88cb974a7d12.xlsx"},{"id":58145484,"identity":"2ccb9b99-6e72-49f5-968e-5d207c1e9c2e","added_by":"auto","created_at":"2024-06-11 18:33:00","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10263,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/ff75079935fe0052438f7f0b.xlsx"},{"id":58145489,"identity":"e543ea54-961a-4179-bc4f-cf6d5bbd9a0f","added_by":"auto","created_at":"2024-06-11 18:33:01","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":99816,"visible":true,"origin":"","legend":"","description":"","filename":"yutinDNAPs2024supplfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4452861/v1/306b43cc7cc18d046031c60e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Jumping DNA polymerases in bacteriophages ","fulltext":[{"header":"Background","content":"\u003cp\u003eViruses with large double-stranded (ds) DNA genomes in the realm \u003cem\u003eDuplodnaviria\u003c/em\u003e share a uniformly conserved structural gene module but vary greatly in their repertoires of DNA replication proteins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Some viruses encode most of the proteins required for DNA replication, whereas others rely (almost) entirely on the replication machinery of the host. Generally, the self-sufficiency of DNA replication correlates with the viral genome size. DNA polymerases (DNAPs) are central components of the viral replication systems that are present in more than half of the available genomes of duplodnaviruses greater than 40kb in size [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. There are four major DNAP families involved in the genome replication in cellular life forms, families A, B, C and D (hereafter PolA-D), with the A, B and C families also being common among DNA viruses. The core catalytic domains of these DNAPs adopt three unrelated folds, namely, (i) the RNA Recognition Motif (RRM), often called the Palm domain (joins the accessory Thumb and Fingers domains) in PolA and PolB, (ii) nucleotidyltransferase Polβ-like fold in PolC, and (iii) the double-psi beta-barrel domain in PolD [\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e–\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In bacteria, PolC is the primary polymerase responsible for the genome replication, whereas PolA is involved in DNA repair processes; PolB is rare in bacteria and is apparently derived from viruses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In archaea, replication is catalyzed by either PolB or PolD, and paralogs of PolB are also involved in repair [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In eukaryotes, almost all processes of DNA synthesis involved in both replication and repair are catalyzed by DNAPs of the PolB family in the nucleus and PolA in mitochondria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent groups of tailed viruses of the class \u003cem\u003eCaudoviricetes\u003c/em\u003e infecting bacteria and archaea encode PolA, PolB or PolC (or no DNAP at all), PolA being the most common, and PolC the rarest [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. All large dsDNA viruses of eukaryotes, in the realms \u003cem\u003eDuplodnaviria\u003c/em\u003e (phylum \u003cem\u003ePeploviricota\u003c/em\u003e) and \u003cem\u003eVaridnaviria\u003c/em\u003e (phylum \u003cem\u003eNucleocytoviricota\u003c/em\u003e), and unassigned class \u003cem\u003eNaldaviricetes\u003c/em\u003e (baculo-like viruses), employ PolB [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Many smaller viruses (with \u0026lt; 50 kb genomes), especially in the realm \u003cem\u003eVaridnaviria\u003c/em\u003e (e.g., polintons, adenoviruses, tectiviruses), replicate with the help of a distinct variety of B family DNAPs, the protein-primed PolB [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], or, less commonly, PolA, also referred to as TV-Pol [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, archaeal viruses exclusively encode family B DNAPs which can be either RNA- or protein-primed [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe DNAPs are essential proteins that are highly conserved within each family, at the sequence and structure levels [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, it came as a surprise that among closely related genomes of phages in the order \u003cem\u003eCrassvirales\u003c/em\u003e, multiple replacements of PolA with PolB and vice versa were observed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similar replacement of replication proteins was detected also among smaller phages of the order \u003cem\u003eVinavirales\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It is particularly notable that in each of these cases, the replacements occurred within otherwise conserved genomic contexts.\u003c/p\u003e \u003cp\u003eIn this work, we aimed to systematically identify and explore cases of between-family DNAP swapping in \u003cem\u003eCaudoviricetes\u003c/em\u003e. We show that DNAPs were swapped repeatedly in the evolution of multiple groups of tailed phages.\u003c/p\u003e \n\n\u003cp\u003e\u003c/p\u003e \n\n "},{"header":"Methods","content":"\u003ch3\u003eDataset of phage genomes and phage genome tree\u003c/h3\u003e\u003cp\u003eGenome-wide relationships between the 18,382 \u003cem\u003eCaudoviricetes\u003c/em\u003e genomes, available in GenBank as of November 2022, were analyzed using reciprocal best hits between viral protein sequences as follows. The set of non-nested ORFs of at least 75 bp was obtained for each virus genome using the NCBI ORFfinder tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Reciprocal best hits for all pairs of genomes (\u003cem\u003eA\u003c/em\u003e,\u003cem\u003eB\u003c/em\u003e), covering at least 50% of the query sequences were identified between the two ORF complements using BLASTP [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Distance between the two genomes was calculated as\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${D}_{A,B}={D}_{B,A}=1-({C}_{A,B}+{C}_{B,A})/({L}_{A}+{L}_{B})$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003eA,B\u003c/em\u003e\u003c/sub\u003e is the total length of the part of genome \u003cem\u003eA\u003c/em\u003e, covered by ORFs that have reciprocal best hits in genome \u003cem\u003eB\u003c/em\u003e and \u003cem\u003eL\u003c/em\u003e\u003csub\u003e\u003cem\u003eA\u003c/em\u003e,\u003c/sub\u003e is the length of genome \u003cem\u003eA\u003c/em\u003e (ditto for \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003eB,A\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eL\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e).\u003c/p\u003e\u003cp\u003eThe tree was reconstructed from the pairwise distance matrix using the FastMe 2.0 program [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and ultrameterized by iteratively balancing subtrees, descending from each internal node.\u003c/p\u003e\u003ch2\u003eIdentification of phage DNA polymerases\u003c/h2\u003e\u003cp\u003ePolA, PolB, PolC reference protein sequences were collected from the NCBI virus database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and from the respective publications, in particular, the PolA sequences were from [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]; PolB sequences were from [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and PolC sequences were from [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/\u003c/span\u003e\u003cspan address=\"https://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Open reading frames (ORFs) from the 18,382 \u003cem\u003eCaudoviricetes\u003c/em\u003e genomes were searched for polymerases using BLASTP with collected reference PolA, PolB, PolC proteins as queries (e-value threshold of 0.0001). The initial set of hits was clustered using MMSEQS2 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] at similarity threshold 0.5; sequences within clusters were aligned using MUSCLE5 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Cluster alignments were iteratively compared to each other using HHSEARCH and aligned using HHALIGN [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Sequences in the final alignments were examined for the presence of the critical catalytic residues. Full-length sequences predicted to be catalytically active were retained for the downstream analysis (see Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for the final set of polymerases, classified as active).\u003c/p\u003e\u003cp\u003eDNAP swapping hotspots were identified by the presence of DNAPs from different families within a subtree of depth 0.15 (corresponding to ~ 1/3rd of the total tree depth). Sister subtrees exhibiting polymerase diversity were grouped into DNAP-swapping clades.\u003c/p\u003e\u003ch3\u003eComparison of phage genomes\u003c/h3\u003e\u003cp\u003ePairwise genome alignments were constructed using Mauve [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and visualized using with Geneious Prime® 2022.1.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneious.com\u003c/span\u003e\u003cspan address=\"https://www.geneious.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Predicted phage proteins were annotated using CDD [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and HHPRED [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003ePhylogenetic analysis of phage proteins\u003c/h2\u003e\u003cp\u003eThe identified viral PolA, PolB, PolC sequences were combined with homologs identified in a collection of completely sequenced bacterial and archaeal genomes downloaded from NCBI Genomes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ftp.ncbi.nlm.nih.gov/genomes/ASSEMBLY_REPORTS/\u003c/span\u003e\u003cspan address=\"https://ftp.ncbi.nlm.nih.gov/genomes/ASSEMBLY_REPORTS/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in November 2021. Sequences of phage DNAPs of the order \u003cem\u003eCrassvirales\u003c/em\u003e were added from [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The protein sequences were aligned using MUSCLE5 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Phylogenetic trees were constructed using IQ-TREE 2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], with the following models chosen according to BIC by the built-in model finder: VT + F + R10 for PolA, Q.pfam + F + R8 for PolB, and VT + F + R5 for PolC, and visualized with MEGA11 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLarge terminase subunits were aligned using MUSCLE5 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]; constrained and unconstrained phylogenetic trees were reconstructed using IQ-TREE 2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] with the automatically selected evolutionary models and compared using the built-in Approximately Unbiased test.\u003c/p\u003e\u003ch2\u003eProtein structure prediction and analysis\u003c/h2\u003e\u003cp\u003eMSAs for divergent family A DNA polymerases identified in this study (divPolA1 and divPolA2) were submitted to a local installation of ColabFold (colabfold_batch with default settings except “–num-models 1 –num-recycle 3”) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, all individual divPolA1 and divPolA2 were modeled with a singularity version of AlphaFold2 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (version 2.2.0 with the following specifications: “--db_preset = full_dbs –model_preset = monomer_ptm –max_template_date = 2022-10-01”) on the high performance cluster BIOWULF at the NIH. All models were compared to a local version of pdb70 (created on December 10, 2021) using Dali [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] to identify closest structures. Structure-guided alignments between representative divPolAs and closest related structures were obtained using the Dali web server [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and key residues were identified. Representative structures modeled with AlphaFold2 (divPolA1 clade5: CAB4155247, clade6: CAB4155247 and divPolA2 AUR84708) were displayed and superimposed with the respective DNAP structures from pdb using ChimeraX [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDNA polymerase diversity in\u003c/strong\u003e \u003cstrong\u003eCaudoviricetes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the analyzed set of 18,382 \u003cem\u003eCaudoviricetes\u003c/em\u003e genomes, we identified 6560 PolA, 2857 PolB, and 947 PolC proteins (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). The \u003cem\u003eCaudoviricetes\u003c/em\u003e genome tree was split into subtrees at the depth of 0.15, roughly corresponding to a genus level (see an example in Supplementary Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/genome_tree/\u003c/span\u003e\u003c/span\u003e). Of the 1,514 subtrees that included more than one virus genome, 563 were found to encode a DNAP, and 8 encoded more than one DNAP. Analysis of the DNAP distribution pattern revealed two distinct types of DNAP heterogeneities within phage subtrees: (i) DNAPs of different families were encoded in closely related viruses, with a single copy in each genome, implying swapping of DNAP genes (6 subtrees), and (ii) the same viral genome encoded two DNAPs of different families (2 subtrees).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA polymerase swapping in\u003c/strong\u003e \u003cstrong\u003eCaudoviricetes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe identified 6 subtrees in the phage genome tree in which the phages encoded DNAPs of different families. Representative pairs of most closely related genomes with different family DNAPs from these subtrees are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. To investigate the provenance of these groups of phages encoding distinct DNAPs, we examined deeper clades in the phage genome tree that included each of the 6 subtrees (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), apart from \u003cem\u003eCrassvirales\u003c/em\u003e for which frequent PolA-PolB swaps have been reported previously [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Clades 1, 2, and 3 consisted of PolA- and PolB-encoding genomes, and clade 4 genomes encompassed PolA and PolC (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The DNAPs from these clades were reciprocally mapped onto the corresponding PolA, PolB, and PolC trees (Supplementary Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Examination of this mapping suggests that, in addition to inter-family DNAP swaps, some intra-family DNAP swaps occurred in the selected clades, that is, PolA or PolB was apparently replaced by a distinct DNAP of the same family on several occasions. Below we discuss each of these clades in detail, in an attempt to reconstruct the evolutionary scenarios.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhage clades displaying DNAP swapping and examples of exchange between closely related genomes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDNAP\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003egenome\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eOrganism\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003egenome length\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eANI or AAI\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eclade\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003etree00180\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMN184886.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eErwinia phage pEp_SNUABM_08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e39% AAI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eClade1; subtrees 00180\u0026ndash;00189\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMN505213.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerratia phage JS26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63971\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003etree00183\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKF626665.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhage Sano\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e39% AAI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e--\u0026ldquo;--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT161385.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eXanthomonas phage FoX4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60418\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003etree00184\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMN536026.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePseudomonas phage vB_Pae-SS2019XI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e38% AAI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e--\u0026ldquo;--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMF959998.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMarinobacter phage PS6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58226\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003etree01348\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMZ477002.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcinetobacter phage Phab24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93604\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e92.72% ANI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eClade2; subtrees 01336\u0026ndash;01351\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMN276049.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcinetobacter phage BS46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94068\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003etree01419\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMG592602.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVibrio phage 1.237.B._10N.261.52.C5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e99.54% ANI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eClade3; subtrees 01419\u0026ndash;01425\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMG592464.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVibrio phage 1.089.O._10N.261.51.F9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59851\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003etree01472\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT601273.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBacillus phage vB_BsuS-Goe12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e99.74% ANI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eClade4; subtrees 01466\u0026ndash;01481\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMT601274.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBacillus phage vB_BsuS-Goe13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126848\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWe sought to validate the intra-clade cross-family swaps of DNAPs using the large subunit of the terminase (TerL) as the reference. The TerL sequences were collected from the genomes with identified DNAPs within each of the clades 1, 2, 3 and 4 and clade-specific TerL phylogenetic trees were constructed. Then, we constructed topologically constrained trees, separating the TerL from genomes encoding different DNAPs (for example, for clade 1, the constraint separated TerL from PolA- and PolB-bearing genomes). Such constrained topologies represent hypothetical phylogenies where DNAPs of different families are not intermixed within the clade histories. The optimal TerL trees satisfying these constraints were compared to the unconstrained trees, in an attempt to falsify the scenario with multiple DNAP swaps. For all clades, the Approximately Unbiased test decisively rejected the constrained topologies (Supplementary Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e), suggesting that multiple DNAP swaps within each clade did occur.\u003c/p\u003e\n\u003cp\u003eClade 1 included 126 genomes from several genera of the family \u003cem\u003eCasjensviridae\u003c/em\u003e (genome size range 50\u0026ndash;70 kb) of which 96 encoded PolA whereas the remaining 30 encoded PolB. The genome tree for this clade is dominated by a distinct group of PolA (A1 in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) in which 3 disjointed PolB branches (B1-B3) and two branches from a separate group of PolA (A2) are embedded. Altogether, comparison of the phage genome tree with the phylogenetic trees of PolA and PolB suggests 8 independent DNAP swaps including both 6 inter-family (PolA to PolB) exchanges and 2 intra-family (A1 to A2) exchanges (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In most of the phage genomes in this clade, the swapped PolA and PolB genes share the same or similar genomic neighborhood (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eClade 2 (subtrees 01336\u0026ndash;01351 in the phage genome tree) unites phages from genera \u003cem\u003ePlaisancevirus\u003c/em\u003e, \u003cem\u003eSaclayvirus\u003c/em\u003e, \u003cem\u003eBarbavirus\u003c/em\u003e, subfamily \u003cem\u003eOunavirinae\u003c/em\u003e, and several unclassified \u003cem\u003eCaudoviricetes\u003c/em\u003e (genome size range 80\u0026ndash;105 kb). The phages in subtree 01347 encode an additional protein with remote sequence and structural similarity to PolA, which we discuss in the next section. Here we address the apparent PolA to PolB swaps in this clade (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). PolBs of Clade 2 are monophyletic (B4 in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), but PolAs come from three distinct branches (A3, A4 and A5 in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e; see Supplementary Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e for the DNAP trees). As in Clade 1, comparison of the phage genome tree with PolA and PolB phylogenetic trees suggests several independent swaps although the direction and the order of these events is difficult to establish.\u003c/p\u003e\n\u003cp\u003eTwo Acinetobacter phages of Clade 2, Phab24 (MZ477002) and BS46 (MN276049), have average nucleotide identity (ANI) of 93%, and yet, encode DNAPs of different families, PolA and PolB, respectively (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Comparison of the genome organization in the vicinity of the DNAP genes (in which we additionally included the corresponding genome region of Acinetobacter phage TaPaz (MZ043613) because its PolB is most similar to PolB of MN276049 (Acinetobacter phage BS46). suggests that, in this case, the primase-helicase gene was replaced along with the DNAP.\u003c/p\u003e\n\u003cp\u003eIn the upper part of each panel, nucleotide sequence similarity between the compared genomes is shown in green or yellow, on the scale from 0 to 100%. Red arrows denote the genomic regions containing the DNAP genes, visualized on the genome maps. The lower parts, show the blow up of the regions containing the DNAP genes. Functionally annotated homologous genes are shown in the same colors. DNAP genes are labeled with their GenBank protein IDs. Grey shading highlights genomic regions with high nucleotide sequence similarity (percent identity indicated). Pink lines connect genes with significant detectable amino acid sequence similarity detected with BLASTP but no significant nucleotide sequence similarity.\u003c/p\u003e\n\u003cp\u003eClade 3 unites subtrees 01419\u0026ndash;01425 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) and includes phages from genera \u003cem\u003eSashavirus\u003c/em\u003e, \u003cem\u003eNonanavirus\u003c/em\u003e, \u003cem\u003eGorganvirus\u003c/em\u003e, and unclassified \u003cem\u003eCaudoviricetes\u003c/em\u003e, with genome size range of 42.5\u0026ndash;62.5 kb. In this clade, a single PolB to PolA swap appears to have occurred within the subtree01419, whereas the basal PolB genes belong to distinct clades (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In this clade, we identified a pair of nearly identical genomes, Vibrio phage 1.237.B._10N.261.52.C5 (MG592602) and Vibrio phage 1.089.O._10N.261.51.F9 (MG592464), that encode different family DNAPs, PolA and PolB, respectively. Pairwise genome comparison shows that the DNAP neighborhood is the only large region with markedly lower similarity between the two genomes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). The DNAP gene is the only one that obviously was replaced but additional rearrangements might have occurred in the adjacent genomic region containing genes encoding uncharacterized small proteins (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\n\u003cp\u003eClade 4 unites subtrees 01466\u0026ndash;1481 including families \u003cem\u003eTybeckvirinae\u003c/em\u003e and \u003cem\u003eAndrewesvirinae\u003c/em\u003e, genera \u003cem\u003eAudreyjarvisvirus\u003c/em\u003e, \u003cem\u003eSpbetavirus\u003c/em\u003e, \u003cem\u003eLatrobevirus\u003c/em\u003e, \u003cem\u003eSextaecvirus\u003c/em\u003e, \u003cem\u003eSlashvirus\u003c/em\u003e, and several unclassified \u003cem\u003eCaudoviricetes\u003c/em\u003e (genome size range 61\u0026ndash;185 kb). In this clade, the phages encode either PolC or PolA. PolC, specifically group C1, is likely to be ancestral in this assemblage of phages (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). This ancestral PolC apparently was replaced with PolA on two independent occasions (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), and furthermore, underwent several intra-family replacements involving PolC variants from groups C2 and C3. We also identified a pair of closely related genomes in this clade with over 99% ANI, Bacillus phage vB_BsuS-Goe12 (MT601273) and Bacillus phage vB_BsuS-Goe13 (MT601274), that encode DNAPs of different families, PolA and PolC, respectively. Comparative genome analysis of this pair of phages revealed an extended segment of dissimilarity suggesting that several replicative genes, including DnaB-like helicase, DnaG-like primase and RecJ-like exonuclease, traveled together with the DNAPs, but the replacement keeps the gene context unchanged, that is, the genes appear to have been replaced \u003cem\u003een bloc\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eWhen the inter-family swaps were found to have occurred on shallow branches of the virus tree, these events typically involved phages that shared bacterial hosts (Supplementary Figure \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eTwo novel phage PolAs\u003c/h2\u003e\n \u003cp\u003eIn addition to the typical DNAPs, we identified two divergent variants of PolA encoded in phage genomes. One of these, denoted divPolA1, is present in a group of 14 phages (Flavobacterium phage vB_FspM_immuto_3-5A and related phages), with genomes in the range of 155\u0026ndash;190 kb that also encode a \u0026lsquo;regular\u0026rsquo; DNAP, either family A, B, or C (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). The conserved arrangement of genes implicated in replication downstream of the divPolA1 gene suggests that divPolA1 is involved in replication (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). Structural prediction for divPolA1 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) showed that it contains a Palm domain in which the main catalytic residues are conserved, whereas the Thumb domain is truncated and the 3\u0026rsquo; exonuclease domain seems to be inactivated, with all three catalytic aspartates replaced (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, divPolA1 most likely retains the DNAP activity whereas the exonuclease activity is lost.\u003c/p\u003e\n \u003cp\u003eAnother diverged PolA variant, divPolA2, was initially identified in 110 genomes of barbaviruses (Rheinheimera phage vB_RspM_Barba18A and related viruses) with genomes of 80\u0026ndash;85 kb, each also encoding a regular PolB. Additional PSI-BLAST searches against the \u003cem\u003eCaudoviricetes\u003c/em\u003e database using barbavirus divPolA2s as queries revealed more divPolA2 proteins, sometimes with two or three paralogs per phage genome (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eUnlike the PolB of these phages, which is embedded within a typical context of replication-related genes, the gene encoding divPolA2 is located in variable gene neighborhood. Structural modeling suggests that divPolA2 contains an active DNAP (Palm) catalytic domain but lacks a Thumb domain homologous to those of any other DNAPs (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eInstead, this protein contains an N-terminal globular domain without detectable similarity to any other known domains that potentially might function as the Thumb. As in the case of divPolA1, the 3\u0026rsquo; exonuclease domain is lacking. Most likely, divPolA2 is not the replicative enzyme of barbaviruses, a role that belongs to PolB. Instead, divPolA2 might be a DNAP involved in repair processes, or an RNA polymerase, given that PolA was co-opted for that function in T7 and related phages [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Of note, structural comparison did not only reveal DNAPs as the top hits for divPolA2, but also DNA-directed RNA polymerases (mitochondrial RNA polymerase (PDB ids: 7a8p, 6ymv, Dali z-score\u0026thinsp;~\u0026thinsp;12) and, with lower z-score (~\u0026thinsp;9), also a viral DNA-directed RNA polymerase from bacteriophage N4 (genus \u003cem\u003eEnquatrovirus\u003c/em\u003e, class \u003cem\u003eCaudoviricetes\u003c/em\u003e) (PDB id: 4ff3). These observations are compatible with the possibility that divPolA2 is actually an RNA polymerase although the N-terminal globular domain of divPolA2 is unrelated to the N-terminal domains of PolA-related RNA polymerases (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTailed viruses of bacteria and archaea that comprise the class \u003cem\u003eCaudoviricetes\u003c/em\u003e in the realm \u003cem\u003eDuplodnaviria\u003c/em\u003e are considered to be the most abundant group of viruses on earth [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Although the virion structures and the core structural proteins are conserved throughout the realm, these viruses greatly differ in their genome size and gene repertoires. In particular, some caudoviricetes encode a (nearly) complete suite of proteins required for replication, whereas others have none, and the entire range of intermediates exists as well [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This variety notwithstanding, more than half of the caudoviricetes encode a DNAP \u0026ndash; the obvious centerpiece of the replication machinery \u0026ndash; that belongs to either A or B, or C family. Generally, the DNAP is a conserved component of the replication apparatus. Unexpectedly, however, in our previous comparative genomic analysis of \u003cem\u003eCrassvirales\u003c/em\u003e (the order of \u003cem\u003eCaudoviricetes\u003c/em\u003e that includes the most abundant viruses identified in the human gut), we found that DNAPs were swapped between closely related phages on multiple occasions, with PolB replacing PolA or vice versa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Intrigued by this observation, we probed a much broader range of phages and report here that multiple DNAP swaps occurred in at least four additional phage groups.\u003c/p\u003e \u003cp\u003eThe DNAP replacements involved either different families, that is, PolA to PolB and vice versa, as well as PolC to PolA, or distinct groups within the same DNAP family. Remarkably, these replacements in each case occurred \u0026ldquo;in situ\u0026rdquo;, without a change in the neighboring gene arrangement. The swap involved either the DNAP gene alone or several adjacent genes encoding other components of the replication machinery, but in each case, the gene replacement appears to have occurred with \u0026ldquo;surgical precision\u0026rdquo;. The genes for proteins involved in replication tend to cluster in viral genomes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and the preservation of their order upon DNAP swapping implies that coregulation of these genes is important for phage reproduction.\u003c/p\u003e \u003cp\u003eThe recurrent DNAP swapping in phage evolution raises intriguing questions on both the molecular mechanisms of these exchanges and the selective forces that could drive them.\u003c/p\u003e \u003cp\u003eThe mechanisms of DNAP swapping remain enigmatic considering the striking precision of these events. Whether or not the phages involved in the swaps are within the range of sequence identity required for homologous recombination, it hardly can contribute to the capture of distantly related genes. Whether the replacing DNAP comes from a prophage integrated in the host cell genome or a coinfecting phage, illegitimate recombination seems to be essential, and the positive selection associated with the swap should be strong enough to provide for the fixation of the rarely emerging precise replacements. In cases where we could pinpoint the intra-family DNAP swaps to a narrow phylogenetic context (that is, between closely related phages), they typically occurred between phages that infect the same host (at least up to the genus level; Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These observations are compatible with the involvement of coinfection, either cotemporaneous or sequential, in DNAP exchanges.\u003c/p\u003e \u003cp\u003eWith respect to the evolutionary forces driving DNAP swapping, it has been shown that multiple defense systems specifically target the phage replication machinery components [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Involvement of at least four types of known defense mechanisms can be suspected. Mutations within DNAP genes have been demonstrated to allow phages to escape restriction by the poorly understood Borvo defense system [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Although the mechanism of Borvo activation remains unclear, it has been suggested that the DNAP structure, its complex with other proteins and/or DNA encompasses molecular patterns that activate Borvo [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Similarly, AbiQ, a type III toxin-antitoxin abortive infection system, was shown to be activated by various phage proteins, with escape mutants localized to a family A DNAP [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Another recent study has similarly shown that mutations in PolB of T-even phages enabled escape from DarTG, a type II toxin-antitoxin system that provides immunity by ADP-ribosylating phage DNA [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Furthermore, pattern recognition systems, in particular, those centered at antivirus STAND ATPases (Avs), have been shown to target conserved viral structural proteins, such as the terminase large subunit and the portal protein [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These viral proteins are conserved at the level of structures even if their sequences diverge relatively fast. The DNAPs, although not universal among tailed phages, unlike terminase and portal, are common and even more highly conserved at the sequence level, and therefore, the existence of multiple pattern recognition systems targeting DNAPs appears likely.\u003c/p\u003e \u003cp\u003eThe high sequence conservation of the DNAPs within each family suggests potential involvement of another type of defense, namely, adaptive immunity mediated by CRISPR systems, and more specifically, primed adaptation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. CRISPR spacers targeting conserved sequences in the DNAP genes are likely to retain complementarity level sufficient for primed adaptation longer than they do in the case of less conserved genes, facilitating acquisition of immunity to the respective phages. Furthermore, existence of yet unknown defense mechanisms targeting DNAPs remains a possibility. An additional or alternative driver of replication module swapping between phages could be the incompatibility of closely related replicons within a coinfected cell, analogous to plasmid incompatibility [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Further study of the notable but not yet well understood phenomenon of DNAP swapping in phages has the potential to reveal unknown facets of interactions between phages and their bacterial hosts as well as conflicts among different phages.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe show in this work that replacement of DNAPs by distantly related or even unrelated ones is common in the evolution of tailed phages of the class \u003cem\u003eCaudovirecetes\u003c/em\u003e. Remarkably, DNAP swapping always occurs \u0026ldquo;in situ\u0026rdquo;, with the organization of the surrounding genes, typically, encoding other proteins involved in phage genome replication being preserved, whether the DNAP gene is the only region of substantial divergence between closely related phage genomes, or the replacement involves several neighboring genes. We hypothesize that although illegitimate recombination is required for replacement of the DNAP genes, selection driving such replacements is strong enough to allow the rare emerging variants with precise insertion of the new sequence to be fixed in the phage population. The factors underlying this selection likely include avoidance of host defense mechanism, such as Borvo, pattern recognition or CRISPR primed adaptation, that target DNAPs. In addition to DNAP swapping, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is based on the analysis of genomes publicly available in GenBank. All other data generated by this analysis are contained in the Supplementary Material or publicly available at https://ftp.ncbi.nih.gov/pub/yutinn/jumping_polymerases_2024/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.Y., P.M., I.T., Y.I.W. and E.V.K. are funded by the Intramural Research Program of the National Institutes of Health (National Library of Medicine).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.Y. and E.V.K. conceptualized the project; N.Y., I.T., and Y.I.W. developed the methodology; N.Y., P.M., Y. I. W., M.K. and E.V.K. analyzed the data; N.Y. and E.V.K. wrote the manuscript; all authors edited and approved the manuscript. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis work utilized the computational resources of the NIH HPC Biowulf cluster (\u003c/em\u003ehttp://hpc.nih.gov\u003cem\u003e).\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKoonin EV, Dolja VV, Krupovic M, Varsani A, Wolf YI, Yutin N, Zerbini FM, Kuhn JH: \u003cstrong\u003eGlobal Organization and Proposed Megataxonomy of the Virus World\u003c/strong\u003e. \u003cem\u003eMicrobiol Mol Biol Rev\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e84\u003c/strong\u003e(2)::e00061-00019.\u003c/li\u003e\n \u003cli\u003eWeigel C, Seitz H: \u003cstrong\u003eBacteriophage replication modules\u003c/strong\u003e. \u003cem\u003eFEMS Microbiol Rev\u0026nbsp;\u003c/em\u003e2006, \u003cstrong\u003e30\u003c/strong\u003e(3):321-381.\u003c/li\u003e\n \u003cli\u003eKazlauskas D, Krupovic M, Venclovas C: \u003cstrong\u003eThe logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res\u0026nbsp;\u003c/em\u003e2016, \u003cstrong\u003e44\u003c/strong\u003e(10):4551-4564.\u003c/li\u003e\n \u003cli\u003eKoonin EV, Krupovic M, Ishino S, Ishino Y: \u003cstrong\u003eThe replication machinery of LUCA: Common origin of DNA replication and transcription\u0026nbsp;\u003c/strong\u003e\u003cem\u003eBMC Biology\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003ein press\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eCzernecki D, Nourisson A, Legrand P, Delarue M: \u003cstrong\u003eReclassification of family A DNA polymerases reveals novel functional subfamilies and distinctive structural features\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e51\u003c/strong\u003e(9):4488-4507.\u003c/li\u003e\n \u003cli\u003eRaia P, Delarue M, Sauguet L: \u003cstrong\u003eAn updated structural classification of replicative DNA polymerases\u003c/strong\u003e. \u003cem\u003eBiochem Soc Trans\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e47\u003c/strong\u003e(1):239-249.\u003c/li\u003e\n \u003cli\u003eKazlauskas D, Krupovic M, Guglielmini J, Forterre P, Venclovas C: \u003cstrong\u003eDiversity and evolution of B-family DNA polymerases\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e48\u003c/strong\u003e(18):10142-10156.\u003c/li\u003e\n \u003cli\u003eSauguet L: \u003cstrong\u003eThe Extended \u0026quot;Two-Barrel\u0026quot; Polymerases Superfamily: Structure, Function and Evolution\u003c/strong\u003e. \u003cem\u003eJ Mol Biol\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e431\u003c/strong\u003e(20):4167-4183.\u003c/li\u003e\n \u003cli\u003eKornberg A, Baker TS: \u003cstrong\u003eDNA Replication\u003c/strong\u003e, 2nd edn. San Francisco: Freeman; 1992.\u003c/li\u003e\n \u003cli\u003eGreci MD, Bell SD: \u003cstrong\u003eArchaeal DNA Replication\u003c/strong\u003e. \u003cem\u003eAnnu Rev Microbiol\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e74\u003c/strong\u003e:65-80.\u003c/li\u003e\n \u003cli\u003eBurgers PM: \u003cstrong\u003ePolymerase dynamics at the eukaryotic DNA replication fork\u003c/strong\u003e. \u003cem\u003eJ Biol Chem\u0026nbsp;\u003c/em\u003e2009, \u003cstrong\u003e284\u003c/strong\u003e(7):4041-4045.\u003c/li\u003e\n \u003cli\u003eBurgers PMJ, Kunkel TA: \u003cstrong\u003eEukaryotic DNA Replication Fork\u003c/strong\u003e. \u003cem\u003eAnnu Rev Biochem\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e86\u003c/strong\u003e:417-438.\u003c/li\u003e\n \u003cli\u003eKrupovic M, Koonin EV: \u003cstrong\u003ePolintons: a hotbed of eukaryotic virus, transposon and plasmid evolution\u003c/strong\u003e. \u003cem\u003eNat Rev Microbiol\u0026nbsp;\u003c/em\u003e2015, \u003cstrong\u003e13\u003c/strong\u003e(2):105-115.\u003c/li\u003e\n \u003cli\u003eSchoenfeld TW, Murugapiran SK, Dodsworth JA, Floyd S, Lodes M, Mead DA, Hedlund BP: \u003cstrong\u003eLateral gene transfer of family A DNA polymerases between thermophilic viruses, aquificae, and apicomplexa\u003c/strong\u003e. \u003cem\u003eMol Biol Evol\u0026nbsp;\u003c/em\u003e2013, \u003cstrong\u003e30\u003c/strong\u003e(7):1653-1664.\u003c/li\u003e\n \u003cli\u003eNasko DJ, Chopyk J, Sakowski EG, Ferrell BD, Polson SW, Wommack KE: \u003cstrong\u003eFamily A DNA Polymerase Phylogeny Uncovers Diversity and Replication Gene Organization in the Virioplankton\u003c/strong\u003e. \u003cem\u003eFront Microbiol\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e9\u003c/strong\u003e:3053.\u003c/li\u003e\n \u003cli\u003eIyer LM, Abhiman S, Aravind L: \u003cstrong\u003eA new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases\u003c/strong\u003e. \u003cem\u003eBiol Direct\u0026nbsp;\u003c/em\u003e2008, \u003cstrong\u003e3\u003c/strong\u003e:39.\u003c/li\u003e\n \u003cli\u003eMakarova KS, Krupovic M, Koonin EV: \u003cstrong\u003eEvolution of replicative DNA polymerases in archaea and their contributions to the eukaryotic replication machinery\u003c/strong\u003e. \u003cem\u003eFrontiers Microbiol\u0026nbsp;\u003c/em\u003e2014, \u003cstrong\u003edoi: 10.3389/fmicb.2014.00354\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003ePrangishvili D, Bamford DH, Forterre P, Iranzo J, Koonin EV, Krupovic M: \u003cstrong\u003eThe enigmatic archaeal virosphere\u003c/strong\u003e. \u003cem\u003eNat Rev Microbiol\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e15\u003c/strong\u003e(12):724-739.\u003c/li\u003e\n \u003cli\u003eYutin N, Benler S, Shmakov SA, Wolf YI, Tolstoy I, Rayko M, Antipov D, Pevzner PA, KooninE.V.: \u003cstrong\u003eAnalysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic features\u0026nbsp;\u003c/strong\u003e\u003cem\u003eNature Communications\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e12\u003c/strong\u003e:1044\u003c/li\u003e\n \u003cli\u003eKrupovic M, Bamford DH: \u003cstrong\u003ePutative prophages related to lytic tailless marine dsDNA phage PM2 are widespread in the genomes of aquatic bacteria\u003c/strong\u003e. \u003cem\u003eBMC Genomics\u0026nbsp;\u003c/em\u003e2007, \u003cstrong\u003e8\u003c/strong\u003e:236.\u003c/li\u003e\n \u003cli\u003eYutin N, Rayko M, Antipov D, Mutz P, Wolf YI, Krupovic M, Koonin EV: \u003cstrong\u003eVaridnaviruses in the Human Gut: A Major Expansion of the Order Vinavirales\u003c/strong\u003e. \u003cem\u003eViruses\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e14\u003c/strong\u003e(9).\u003c/li\u003e\n \u003cli\u003eAltschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: \u003cstrong\u003eGapped BLAST and PSI-BLAST: a new generation of protein database search programs\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res\u0026nbsp;\u003c/em\u003e1997, \u003cstrong\u003e25\u003c/strong\u003e(17):3389-3402.\u003c/li\u003e\n \u003cli\u003eLefort V, Desper R, Gascuel O: \u003cstrong\u003eFastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program\u003c/strong\u003e. \u003cem\u003eMol Biol Evol\u0026nbsp;\u003c/em\u003e2015, \u003cstrong\u003e32\u003c/strong\u003e(10):2798-2800.\u003c/li\u003e\n \u003cli\u003eKeown RA, Dums JT, Brumm PJ, MacDonald J, Mead DA, Ferrell BD, Moore RM, Harrison AO, Polson SW, Wommack KE: \u003cstrong\u003eNovel Viral DNA Polymerases From Metagenomes Suggest Genomic Sources of Strand-Displacing Biochemical Phenotypes\u003c/strong\u003e. \u003cem\u003eFront Microbiol\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e13\u003c/strong\u003e:858366.\u003c/li\u003e\n \u003cli\u003eDorawa S, Werbowy O, Plotka M, Kaczorowska AK, Makowska J, Kozlowski LP, Fridjonsson OH, Hreggvidsson GO, Aevarsson A, Kaczorowski T: \u003cstrong\u003eMolecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity\u003c/strong\u003e. \u003cem\u003eInt J Mol Sci\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e23\u003c/strong\u003e(14).\u003c/li\u003e\n \u003cli\u003eTiminskas K, Venclovas C: \u003cstrong\u003eNew insights into the structures and interactions of bacterial Y-family DNA polymerases\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e47\u003c/strong\u003e(9):4393-4405.\u003c/li\u003e\n \u003cli\u003eSteinegger M, Soding J: \u003cstrong\u003eMMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets\u003c/strong\u003e. \u003cem\u003eNat Biotechnol\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e35\u003c/strong\u003e(11):1026-1028.\u003c/li\u003e\n \u003cli\u003eEdgar RC: \u003cstrong\u003eMuscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny\u003c/strong\u003e. \u003cem\u003eNat Commun\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e13\u003c/strong\u003e(1):6968.\u003c/li\u003e\n \u003cli\u003eZimmermann L, Stephens A, Nam SZ, Rau D, Kubler J, Lozajic M, Gabler F, Soding J, Lupas AN, Alva V: \u003cstrong\u003eA Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core\u003c/strong\u003e. \u003cem\u003eJ Mol Biol\u0026nbsp;\u003c/em\u003e2018, \u003cstrong\u003e430\u003c/strong\u003e(15):2237-2243.\u003c/li\u003e\n \u003cli\u003eDarling AC, Mau B, Blattner FR, Perna NT: \u003cstrong\u003eMauve: multiple alignment of conserved genomic sequence with rearrangements\u003c/strong\u003e. \u003cem\u003eGenome Res\u0026nbsp;\u003c/em\u003e2004, \u003cstrong\u003e14\u003c/strong\u003e(7):1394-1403.\u003c/li\u003e\n \u003cli\u003eMarchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eCDD/SPARCLE: functional classification of proteins via subfamily domain architectures\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e45\u003c/strong\u003e(D1):D200-D203.\u003c/li\u003e\n \u003cli\u003eSoding J: \u003cstrong\u003eProtein homology detection by HMM-HMM comparison\u003c/strong\u003e. \u003cem\u003eBioinformatics\u0026nbsp;\u003c/em\u003e2005, \u003cstrong\u003e21\u003c/strong\u003e(7):951-960.\u003c/li\u003e\n \u003cli\u003eMinh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, Lanfear R: \u003cstrong\u003eIQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era\u003c/strong\u003e. \u003cem\u003eMol Biol Evol\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e37\u003c/strong\u003e(5):1530-1534.\u003c/li\u003e\n \u003cli\u003eTamura K, Stecher G, Kumar S: \u003cstrong\u003eMEGA11: Molecular Evolutionary Genetics Analysis Version 11\u003c/strong\u003e. \u003cem\u003eMol Biol Evol\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e38\u003c/strong\u003e(7):3022-3027.\u003c/li\u003e\n \u003cli\u003eMirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M: \u003cstrong\u003eColabFold: making protein folding accessible to all\u003c/strong\u003e. \u003cem\u003eNat Methods\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e19\u003c/strong\u003e(6):679-682.\u003c/li\u003e\n \u003cli\u003eJumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eHighly accurate protein structure prediction with AlphaFold\u003c/strong\u003e. \u003cem\u003eNature\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e596\u003c/strong\u003e(7873):583-589.\u003c/li\u003e\n \u003cli\u003eHolm L: \u003cstrong\u003eDALI and the persistence of protein shape\u003c/strong\u003e. \u003cem\u003eProtein Sci\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e29\u003c/strong\u003e(1):128-140.\u003c/li\u003e\n \u003cli\u003eHolm L: \u003cstrong\u003eDali server: structural unification of protein families\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e50\u003c/strong\u003e(W1):W210-W215.\u003c/li\u003e\n \u003cli\u003ePettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE: \u003cstrong\u003eUCSF ChimeraX: Structure visualization for researchers, educators, and developers\u003c/strong\u003e. \u003cem\u003eProtein Sci\u0026nbsp;\u003c/em\u003e2021, \u003cstrong\u003e30\u003c/strong\u003e(1):70-82.\u003c/li\u003e\n \u003cli\u003eSuttle CA: \u003cstrong\u003eViruses in the sea\u003c/strong\u003e. \u003cem\u003eNature\u0026nbsp;\u003c/em\u003e2005, \u003cstrong\u003e437\u003c/strong\u003e(7057):356-361.\u003c/li\u003e\n \u003cli\u003eMushegian AR: \u003cstrong\u003eAre There 10(31) Virus Particles on Earth, or More, or Fewer?\u003c/strong\u003e \u003cem\u003eJ Bacteriol\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e202\u003c/strong\u003e(9).\u003c/li\u003e\n \u003cli\u003eStokar-Avihail A, Fedorenko T, Hor J, Garb J, Leavitt A, Millman A, Shulman G, Wojtania N, Melamed S, Amitai G\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eDiscovery of phage determinants that confer sensitivity to bacterial immune systems\u003c/strong\u003e. \u003cem\u003eCell\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e186\u003c/strong\u003e(9):1863-1876 e1816.\u003c/li\u003e\n \u003cli\u003eMillman A, Melamed S, Leavitt A, Doron S, Bernheim A, Hor J, Garb J, Bechon N, Brandis A, Lopatina A\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e: \u003cstrong\u003eAn expanded arsenal of immune systems that protect bacteria from phages\u003c/strong\u003e. \u003cem\u003eCell Host Microbe\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e30\u003c/strong\u003e(11):1556-1569 e1555.\u003c/li\u003e\n \u003cli\u003eHuiting E, Bondy-Denomy J: \u003cstrong\u003eDefining the expanding mechanisms of phage-mediated activation of bacterial immunity\u003c/strong\u003e. \u003cem\u003eCurr Opin Microbiol\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e74\u003c/strong\u003e:102325.\u003c/li\u003e\n \u003cli\u003eSamson JE, Belanger M, Moineau S: \u003cstrong\u003eEffect of the abortive infection mechanism and type III toxin/antitoxin system AbiQ on the lytic cycle of Lactococcus lactis phages\u003c/strong\u003e. \u003cem\u003eJ Bacteriol\u0026nbsp;\u003c/em\u003e2013, \u003cstrong\u003e195\u003c/strong\u003e(17):3947-3956.\u003c/li\u003e\n \u003cli\u003eLeRoux M, Srikant S, Teodoro GIC, Zhang T, Littlehale ML, Doron S, Badiee M, Leung AKL, Sorek R, Laub MT: \u003cstrong\u003eThe DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA\u003c/strong\u003e. \u003cem\u003eNat Microbiol\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e7\u003c/strong\u003e(7):1028-1040.\u003c/li\u003e\n \u003cli\u003eGao LA, Wilkinson ME, Strecker J, Makarova KS, Macrae RK, Koonin EV, Zhang F: \u003cstrong\u003eProkaryotic innate immunity through pattern recognition of conserved viral proteins\u003c/strong\u003e. \u003cem\u003eScience\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e377\u003c/strong\u003e(6607):eabm4096.\u003c/li\u003e\n \u003cli\u003eKibby EM, Conte AN, Burroughs AM, Nagy TA, Vargas JA, Whalen LA, Aravind L, Whiteley AT: \u003cstrong\u003eBacterial NLR-related proteins protect against phage\u003c/strong\u003e. \u003cem\u003eCell\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e186\u003c/strong\u003e(11):2410-2424 e2418.\u003c/li\u003e\n \u003cli\u003eJackson SA, Birkholz N, Malone LM, Fineran PC: \u003cstrong\u003eImprecise Spacer Acquisition Generates CRISPR-Cas Immune Diversity through Primed Adaptation\u003c/strong\u003e. \u003cem\u003eCell Host Microbe\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e25\u003c/strong\u003e(2):250-260 e254.\u003c/li\u003e\n \u003cli\u003eShiriaeva AA, Kuznedelov K, Fedorov I, Musharova O, Khvostikov T, Tsoy Y, Kurilovich E, Smith GR, Semenova E, Severinov K: \u003cstrong\u003eHost nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system\u003c/strong\u003e. \u003cem\u003eSci Adv\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e8\u003c/strong\u003e(47):eabn8650.\u003c/li\u003e\n \u003cli\u003eIgler C, Huisman JS, Siedentop B, Bonhoeffer S, Lehtinen S: \u003cstrong\u003ePlasmid co-infection: linking biological mechanisms to ecological and evolutionary dynamics\u003c/strong\u003e. \u003cem\u003ePhilos Trans R Soc Lond B Biol Sci\u0026nbsp;\u003c/em\u003e2022, \u003cstrong\u003e377\u003c/strong\u003e(1842):20200478.\u003c/li\u003e\n \u003cli\u003ePilosof S: \u003cstrong\u003eConceptualizing microbe-plasmid communities as complex adaptive systems\u003c/strong\u003e. \u003cem\u003eTrends Microbiol\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e31\u003c/strong\u003e(7):672-680.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Evolution of Viruses, DNA polymerases, Bacterial antivirus defense, Horizontal gene transfer","lastPublishedDoi":"10.21203/rs.3.rs-4452861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4452861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eViruses with double-stranded (ds) DNA genomes in the realm \u003cem\u003eDuplodnaviria\u003c/em\u003e share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order \u003cem\u003eCrassvirales\u003c/em\u003e encode DNAPs of different families.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGroups of phages in which DNAP swapping likely occurred were identified as subtrees of a defined depth in a comprehensive evolutionary tree of tailed bacteriophages that included phages with DNAPs of different families. The DNAP swaps were validated by constrained tree analysis that was performed on phylogenetic tree of large terminase subunits, and the phage genomes encoding swapped DNAPs were aligned using Mauve. The structures of the discovered unusual DNAPs were predicted using AlphaFold2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe identified four additional groups of tailed phages in the class \u003cem\u003eCaudoviricetes\u003c/em\u003e in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs \u0026ldquo;in situ\u0026rdquo;, without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage replication. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eReplacement of the DNAP gene by one encoding a DNAP of a different family occurred on many independent occasions during the evolution of different families of tailed phages, in some cases, resulting in very closely related phages encoding unrelated DNAPs. DNAP swapping was likely driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility.\u003c/p\u003e","manuscriptTitle":"Jumping DNA polymerases in bacteriophages ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 18:32:55","doi":"10.21203/rs.3.rs-4452861/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T10:24:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-07T00:12:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-03T14:37:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234659864970691288127445387144382399453","date":"2024-06-26T18:51:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305675428581851972029162295405611038758","date":"2024-06-25T09:09:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-03T22:28:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160683747519588128077563668087191168656","date":"2024-05-28T01:26:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-27T10:34:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-26T23:33:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-26T22:56:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2024-05-21T07:17:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd5517cb-a771-4851-bcf8-b4edb33b49a4","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T16:03:19+00:00","versionOfRecord":{"articleIdentity":"rs-4452861","link":"https://doi.org/10.1186/s12985-024-02482-z","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2024-08-26 15:57:47","publishedOnDateReadable":"August 26th, 2024"},"versionCreatedAt":"2024-06-11 18:32:55","video":"","vorDoi":"10.1186/s12985-024-02482-z","vorDoiUrl":"https://doi.org/10.1186/s12985-024-02482-z","workflowStages":[]},"version":"v1","identity":"rs-4452861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4452861","identity":"rs-4452861","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}