Patent ID: 12188057

EXAMPLES

The following examples are provided to illustrate the invention, but are not intended to limit the scope of the same. Said examples are based on assays carried out by the inventors and show the efficient primer-independent replication activity of the piPolB of SEQ ID NO: 2, as well as its translesion synthesis capacity.

Example 1. A Novel Major Group of Family B DNA Polymerases

PSI-BLAST searches against the RefSeq bacterial genome database at NCBI seeded with the sequence of experimentally characterized pPolB from bacteriophage Bam35 (NP_943751) retrieved numerous hits to PolBs. These could be categorized into two groups: (i) highly significant hits (38-99% sequence identity) to pPolBs encoded within genomic contigs related to bacteriophages Bam35 (family Tectiviridae) and CD29 (subfamily Picovirinae, family Podoviridae); (ii) hits to highly divergent pPolB homologs encoded within chromosomes and several plasmids from widely diverse bacteria, such as Firmicutes, Actinobacteria and Proteobacteria. The latter proteins displayed ˜16-20% sequence identity to the pPolB of Bam35. Nevertheless, analysis of multiple sequence alignments of these putative divergent DNAPs showed that all of them contain the TPR1 and TPR2 subdomains, a hallmark of pPolBs, and the active site residues of the exonuclease and DNA polymerase domains of PolBs are conserved, albeit with notable variations within the KxY and PolC motifs. The PolC motif (DTD) is almost universally conserved in PolBs and contains two catalytic aspartic acid residues required for protein activity. In the present invention, it was noticed that in all members of the novel piPolB group, the first of the two aspartates within the PolC motif is substituted for a threonine residue (TTD). Notably, some archaeal pPolBs also show variation within this motif, but none of these proteins has been experimentally characterized.

Additional searches seeded with representative sequences of the novel PolB group from proteobacteria, such asEscherichia coli(KDU42669) orRhodobacterales bacteriumY41 (WP_008555115), yielded significant hits to several homologs encoded by pCRY1-like circular mitochondrial plasmids. Notably, the latter plasmids are distinct from the extensively studied linear mitochondrial plasmids which encode pPolBs (see below). Sequence analysis of the mitochondrial proteins confirmed their close similarity to the divergent group of bacterial PolBs.

To gain further understanding on the relationship between the newly discovered group of DNAPs and pPolBs, a maximum likelihood phylogenetic analysis of representative sequences from all known clades of pPolBs, including bacterial, archaeal and eukaryotic viruses, casposons, polintons, as well as eukaryotic cytoplasmic and mitochondrial linear plasmids, was performed. In the phylogenetic tree rooted with rPolB sequences, all previously characterized pPolBs formed a well-supported monophyletic clade, with a branching pattern consistent with previous phylogenetic analyses. The new DNAPs formed a distinct, well-supported clade, which in the present invention was denoted “piPolB” (see below), separated from all other pPolBs, suggesting that it has diverged early in the evolution of PolBs. Thus, piPolB represents the third major group of PolBs, besides rPolBs and pPolBs.

Within the piPolB clade, there are two major groups, which are roughly congruent with the bacterial taxonomy. The first one predominantly includes sequences from Actinobacteria and several orders of Firmicutes, namely Bacillales, Lactobacillales and Clostridiales. The second group contains sequences from different classes of Proteobacteria, namely Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. Notably, the latter group also includes piPolBs from circular mitochondrial plasmids which cluster with sequences from alphaproteobacteria. Although the latter clustering is not strongly supported in this analysis, this observation is most consistent with a possibility that piPolBs were imported into eukaryotes together with the alphaproteobacterial symbiont at the origin of mitochondria. Thus, piPolBs appear to have coevolved with their hosts for an extended period, several potential cases of horizontal gene transfer notwithstanding.

Example 2. piPolBs are Encoded within a Novel Group of Self-Replicating Elements

Genomic context analysis provided compelling evidence that the majority of piPolBs are encoded within MGEs integrated into bacterial chromosomes. Unlike casposons and polintons which integrate into the genome using Cas1-like endonucleases and retrovirus-like family integrases, respectively, the vast majority of piPolB-carrying MGEs encode integrases of the tyrosine recombinase (Y-integrase) superfamily. Some of the elements carry additional copies of Y-integrases or integrases/invertases of the serine recombinase superfamily. Nevertheless, several bacterial and all mitochondrial piPolB homologs are encoded by extrachromosomal, rather than integrated plasmids and, accordingly, lack the integrase genes, suggesting that integration into the chromosome is optional for these elements. Hence, in the present invention, all these new bacterial and mitochondrial elements were referred to as pipolins (for piPolB-encoding mobile genetic elements).

MGE integration leaves a molecular mark on the cellular chromosome which manifests in the form of direct repeats, corresponding to the left and right attachment sites (attL and attR), which flank the integrated element. The Y-integrases typically catalyze recombination between homologous sites present on the cellular genome and the circular dsDNA molecules of MGEs. Thorough analysis of the piPolB-encompassing genomic regions, allowed to define the precise integration sites for many pipolins from diverse bacterial taxa. The vast majority of integrations occurred within tRNA genes, as is common for bacterial and archaeal MGEs employing Y-integrases. Some bacteria carry more than one pipolin. For instance,Vibrio vulnificusgenome contains two related piPolB-encoding elements inserted into different tRNA genes.

Comparative genomic analysis of pipolins showed that they form groups which are generally consistent with the phylogeny of the piPolBs. The similarity between elements from distantly related hosts is limited to the piPolB and, to a lesser extent, Y-integrase genes. Besides piPolB and integrases, pipolins often encode excisionases, which assist in excision of integrated MGEs; components of type I and type II restriction modification systems; and various components of the plasmid mobilization machinery. In addition, the less conserved genes found in pipolins encode different DNA-binding proteins with ribbon-helix-helix, zinc-finger or helix-turn-helix motifs, but also histone-like H-NS chromatin proteins, various nucleases and toxin-antitoxin systems. None of the elements encodes virus-specific proteins. By contrast, the pangenome of pipolins consists of various genes typical of plasmids. Indeed, protein BLAST (BLASTP) analysis shows that most of the pipolin genes are conserved in various unrelated plasmids. Consistent with this assertion, four of the bacterial and five mitochondrial piPolBs are encoded by circular plasmids. Notably, the mitochondrial plasmids carry no other genes than those encoding piPolB, suggesting that following the introduction of a mitochondrial ancestor into a proto-eukaryotic host, the MGEs underwent reductive evolution, losing all genes except for the piPolB.

Although piPolB is the only DNA replication-associated protein conserved in all pipolins, some elements encode putative helicases of superfamilies 1 and 2; 3′-5′ exonucleases; uracil-DNA glycosylases; ribonucleases H; and an Orc1/Cdc6-like AAA+ATPase. Unlike pPolB-encoding plasmids and viruses which, as a rule, have linear genomes, pipolins represent circular dsDNA molecules and thus the protein-priming mechanism is unlikely to be applicable. The overwhelming majority (94%) of dsDNA viruses encoding RNA-primed DNAPs also encode their own primases. By contrast, none of the pipolins possesses genes for recognizable primases, raising questions regarding the priming mechanism.

Collectively, results of the phylogenetic and comparative genomic analyses underscore the uniqueness of piPolBs and pipolins, which may be considered as the third major superfamily of self-replicating MGEs, next to polintons and casposons.

Example 3. Pipolin DNA Polymerase is a Proficient Replicase

To verify whether piPolBs were indeed active DNAPs, a representative enzyme fromE. coli3-373-03-S1_C2 pipolin (SEQ ID NO: 2) was chosen and its recombinant form was purified. The synthetic and degradative activities of this protein in a primer extension assay were first analyzed, in the presence of increasing concentrations of dNTPs (FIG.1A, lanes 4-9). As expected, only degradation products could be detected in the absence of dNTPs. However, addition of dNTPs resulted in a switch from exonucleolysis to polymerization activity, indicating that both activities are coordinated. Protein variants with deficient polymerization (D368A,FIG.1A, lanes 10-11 andFIG.7A) or exonuclease (D59A/E61A,FIG.7A) activities, confirmed that 5′-3′ synthetic and 3′-5′ degradative capacities are intrinsic to the recombinant purified piPolB. The presence of proficient DNA polymerization activity in piPolB confirms that only the second carboxylate moiety in the PolC motif is required for metal coordination, in agreement with the previous suggestions that the first conserved aspartate residue in the PolC motif in the pPolB and rPolB groups has a structural rather than catalytic role.

To further characterize the DNA polymerization activity of piPolB, the insertion preference for Watson-Crick base pairs was analyzed using the piPolB exonuclease-deficient variant D59A/E61A. As shown inFIG.1B, insertion of the correct nucleotide could be detected at approximately 1000-fold lower dNTP concentration compared with the incorrect dNTP, indicating a very strong preference for the insertion of the complementary nucleotide. These results confirm that piPolB of pipolins is an efficient and faithful DNA polymerase, as would be expected from a replicative family B DNAP.

Example 4. PiPolB is Endowed with Intrinsic Translesion Synthesis Across DNA Containing Non-Bulky Nucleotides Analogs

Abasic (AP) sites constitute the most common DNA lesion that may arise from spontaneous depurination but also occur as intermediates in base excision repair. A prevailing model is that high-fidelity replicative DNAPs are unable to replicate through such lesions in the DNA, leading to stalled replication and subsequent triggering of DNA damage tolerance mechanisms, involving specialized DNAPs that can bypass the DNA damage by translesion synthesis (TLS). Additionally, recent works reported examples of TLS by cellular and viral replicases from families A, B or C during processive genome replication. Thus, to ascertain whether piPolB was able to replicate damaged templates, it was first analyzed primer extension opposite a tetrahydrofuran (THF) moiety, a stable analog of an abasic site, in the first template nucleotide position. As shown inFIG.2A, piPolB is able to insert the first nucleotide and extend the primer beyond the THF (lines 4-9) whereas, as expected, CD29DNAP only gave rise to negligible replication (lines 2-3). The bypass capacity often depends on the sequence context and is counteracted by the proofreading activity. However, piPolB TLS capacity does not seem to be affected by the template sequence context (FIG.7A). A minor band of partial product at the lesion site (16-mer) could be detected, suggesting that, as shown previously for the Bam35 DNAP, elongation of the primer beyond the abasic site is a limiting step in the TLS by piPolB, despite the fact that replication of both, undamaged and damaged oligonucleotide templates can be processive (FIG.7B).

It was then analyzed the incorporation preference opposite to the THF site. Using the exonuclease deficient variant D59A/E61A, it was found that piPolB preferentially inserts purines over pyrimidines (FIG.2B, lanes 3-6), in the preference order A>G>T>C, in agreement with the so-called “A rule” previously described for many DNAPs. The TLS by DNAPs may occur via a misalignment mechanism, resulting in a one or two nucleotide deletion and, accordingly, a shorter DNA product.FIGS.2A and7Ashow that the final product synthesized by piPolB reached the full product length using both damaged and undamaged substrates, suggesting that, instead of a misalignment mechanism, the piPolB can insert and further elongate a nucleotide opposite to the abasic site. To verify this mechanism, it was monitored step-by-step polymerization in a primer extension assay in the presence of different dNTP combinations (FIG.2B, lanes 7-11). In particular, it was provided dATP in combination with another single dNTP (dTTP, dCTP and dGTP, lanes 7-9, respectively). Whereas insertion opposite to the THF was detected in all cases, only the combination dATP and dTTP (AT, lane 7) allowed primer extension beyond the abasic site, giving rise to a product that corresponds with the 19-mer marker (FIG.2B, lane M), indicating accurate replication of the template up to this position (see substrate scheme above the gel). Consistently, the presence of dATP, dTTP and dGTP (ATG, lane 10) allowed the copy of the template up to the 22-mer product length, and only when the four dNTPs were provided, the full-length replication product could be detected (lane 11). Taken together, these results indicate that TLS capacity of piPolB preferably inserts an A opposite to the abasic sites and subsequently elongates the primer processively without introducing frameshift mutations.

In order to obtain a more comprehensive understanding of the piPolB-mediated DNA replication and TLS performance, it was analyzed the abasic site bypass with different metal cofactors and replication-blocking DNA damages. As shown inFIG.2C, abasic site TLS in the presence of manganese ions was more efficient at lower dNTPs concentrations (lanes 19-22 vs. 8-11), in agreement with previous reports on other PolBs. It was noted that replication of undamaged template required higher dNTPs concentration in the presence of manganese ions (lanes 14-17) when compared with the magnesium-triggered reactions (lanes 3-6). The template specificity of piPolB TLS capacity with substrates containing thymine-glycol (Tg) oxidized base and cyclobutane thymine dimers (T:T), was next explored. Tg is the most common oxidative product of thymine and presents a strong block for DNA synthesis by most replicative DNAPs. On the other hand, T:T arises upon exposure to ultraviolet light radiation and constitutes a particularly sharp hindrance for most DNAPs because the covalent linkage of two adjacent nucleobases prevents a kink in the DNA backbone that normally delivers one template base at a time to the polymerase active site. Results indicate that piPolB was able to bypass thymine-glycol (Tg) oxidized base in the presence of magnesium ions (FIG.2D, lanes 5-8). Interestingly, however, primer extension beyond the damage was less efficient, because the 16-mer pause is stronger than in the case of the THF-containing template (lanes 8-11 in panel C vs. lanes 5-7 in panel D). In line with the impairment in processive primer extension beyond the damage, manganese ions apparently did not stimulate the TLS. On the contrary, Tg bypass was reduced in the presence of this metal cofactor (lanes 9-10). In the case of T:T, insertion of only 1 or 2 nucleotides opposite to the damage could be detected, either with magnesium or manganese ions (lanes 12-14 and 15-17, respectively). In conclusion, piPolB has an efficient TLS capacity that allows it to bypass abasic sites and oxidative base modifications but is unable to overcome bulkier modifications such as T:T, likely because this damage induces major structural changes in the DNA helix that strongly obstructs DNA replication.

Example 5. Primer-Independent DNA Replication

To gain insights into the processivity of the DNA replication by piPolB, it was performed singly primed ssDNA rolling circle replication assays using M13 DNA as a template and resolved the products by alkaline denaturing electrophoresis. Due to its high processivity, coupled with strand-displacement capacity, CD29DNAP was able to synthesize very large ssDNA fragments under these conditions (FIG.3A, lane 1). By contrast, piPolB gave rise to a smeared signal of replication products spanning 0.5 to 10 kb, with apparent peak at about 3 kb (FIG.3A, lanes 3-6), which indicates that piPolB is not as processive as Φ29DNAP and thus generates shorter DNA fragments. However, the maximal product length obtained with piPolB remained similar even at 20-fold lower enzyme concentration, suggesting that it is a processive DNA replicase. A considerable portion of replication products was larger than the M13 DNA, suggesting that piPolB DNA replication is coupled with strand displacement. The latter activity was subsequently confirmed using an oligonucleotide template/primer substrate with a 5-nt gap (FIG.8).

Strikingly, a very similar replication pattern was detected regardless of whether the M13 was primed or not (FIG.3A, lanes 10-13). By contrast, as expected, Φ29DNAP was unable to synthesize any product in the absence of a primer (lane 9). When the same samples were loaded on a non-denaturing agarose gel (FIG.3B), the replication product appeared as a single band that corresponded to the expected M13 unit length, suggesting that the single-stranded DNA products detected in alkaline denaturing electrophoresis gel are M13 replication products. Consistently, no product could be detected in the absence of input DNA template (lanes 16-19), ruling out the replication of possible contaminant DNA traces in any of the polymerase variants. Similarly, no product was obtained when the reactions were performed with the D368A variant deficient for polymerization activity (lanes 8 and 15). Notably, the fragments detected with the D59A/E61A mutant were slightly smaller (by <0.5 kb) than with the wild type enzyme (FIG.3Alanes 7 and 14), presumably because exonuclease deficiency gives rise to the accumulation of replication mistakes that may result in the impairment of strand-displacement or processivity. These results further confirm that M13 DNA replication, with or without the added primer, is intrinsic to piPolB. De novo DNA synthesis on non-primed M13 DNA could be detected using both, magnesium or manganese ions, as cofactors (FIG.3C, lanes 2-3 and 8-9), albeit with a somewhat higher intensity of total replication product with manganese ions. However, replication was not detected when deoxyribonucleotides were substituted with ribonucleotides (lanes 5-6 and 11-12), as expected for a family B DNAP that contains the conserved tyrosine steric gate. Smaller DNA fragments were detected with the wild type polymerase that might be products of the exonucleolytic degradation (lane 8).

To investigate a possible sequence requirement for de novo initiation of DNA replication, assays using a single-stranded homolymeric poly-dT 33-mer as a template were performed. As shown inFIG.3D, piPolB could replicate this template, suggesting that there might not be a requirement for a specific template sequence under the conditions used. DNA replication in the presence of the complementary dATP gave rise to large DNA products and a laddered pattern indicating that replication started de novo, with the synthesis of short primers. This laddered pattern could correspond either to a distributive replication or alternative initiation positions throughout the template. Replication products obtained using the exonuclease-deficient piPolB where overall shorter, suggesting a processivity impairment, as found in the case of M13 replication (FIG.9, lanes 8-10 vs. 11-13). Using this short, homopolymeric substrate, DNA primer synthesis was negligible with magnesium ions (FIG.9, lanes 2-7 vs. 8-13), underlining the higher efficiency of manganese as a cofactor for DNA priming, in agreement with the previous results with M13 DNA (FIG.3C).

Interestingly, when all dNTPs were added, generation of large DNA products was somewhat reduced (FIG.3D, lane 6) and, if dATP was reduced to the labeled nucleotide (16 nM compared with 100 μM of the non-labeled, lane 7), replication products were negligible, which suggests that formation of correct Watson-Crick base pairs is required for replication initiation.

Collectively, these results indicate that piPolB fromE. coli3-373-03_S1_C2 pipolin is able to initiate and perform DNA replication of circular and linear templates in the absence of pre-existing primers or additional protein factors. Furthermore, replication of homopolymeric DNA substrates suggests that, contrary to canonical DNA primases from the archaeo-eukarotic primase (AEP) superfamily, piPolB DNA priming capacity does not rely on a specific template sequence.

Example 6. De Novo Synthesis of DNA Primers

To further confirm that piPolB is able to synthesize DNA de novo, it was performed M13 ssDNA replication using γ32P-ATP as a labeled nucleotide. Thus, only newly synthesized DNA fragments would incorporate the radioactive label. As shown inFIG.4A, small DNA fragments (up to 4-5 nucleotides in length) were generated in a distributive manner by wild type and exonuclease deficient piPolBs, but not by the D368A variant. Again, this reaction was considerably more efficient in the presence of manganese ions than with magnesium ions (lanes 1-8 vs. 9-16). Furthermore, the products were only detected in the presence of dNTPs but not with NTPs (not shown). Instead of the large DNA fragments detected in the assays described above (FIG.3A), only di- and trinucleotide primers were observed, which may be abortive initiation products resulting from the incorporation of a ribonucleotide (rather than dNTP) as a terminal 5′ nucleotide.

The use of high resolution PAGE allowed to identify alternative di- and trinucleotide primers with similar intensity, suggesting that DNA synthesis initiation by piPolB does not require a specific template sequence. In line with this, when each dNTP was provided separately (FIG.4B), the reaction was clearly stimulated by dGTP, in the presence of either magnesium or manganese ions (lanes 4 and 14) and, to a lesser extent, by dCTP and dTTP, either alone or in combination with other deoxyribonucleotides. The fact that A-dG dinucleotide was the most efficiently synthetized initiation product is in agreement with the observation that pyrimidines are the preferential template substrates for the priming reaction by most DNA primases. In line with these results, single nucleotide changes in the poly-dT homopolymer substrate did not substantially change the efficiency of de novo DNA synthesis (FIG.10), although short di- and tri-nucleotides could be detected when one or two Cs were included in the template sequence, even at the 5′ end of the template molecule (lanes 7 and 12). Taken together, these results demonstrate that piPolB is able to initiate de novo DNA primer synthesis without a strong requirement for specific template sequence.

Example 7. An Invariable Lysine Plays a Role in TLS and Primer Synthesis Activities

PolBs contain a conserved KxY motif within a β-strand in the palm domain involved in stabilization of the primer terminus. It was hypothesized that structural adaptations of this motif or nearby residues would be required for stable binding of a nucleoside triphosphate at the 5′-side of the nascent primer to allow dinucleotide formation. Indeed, analysis of the multiple sequence alignment showed that the piPolBs lack the canonical KxY motif and instead contain an alternative conserved sequence KTRG (SEQ ID NO: 46). An additional KX2pattern within an N-terminal extension of the same β-strand is also highly conserved in piPolB homologs, defining an extended KX2-X3-10-KTRG motif (SEQ ID NO: 1). In the representative enzyme of SEQ ID NO: 2 tested herein, X2is H and X3-10is SEQ ID NO: 5, i. e. SEQ ID NO: 1 is SEQ ID NO: 85, and this motif KX2-X3-10-KTRG corresponds to positions 613 to 626 of this sequence. Thus, alanine variants of these K613, H614, K623 and R625 residues were generated. In agreement with a putative role in primer terminus stabilization, K623A and R625A variants had impaired primer extension capacity (FIG.11A) and primer synthesis beyond the dinucleotide formation (FIG.11B). On the other hand, K613A and H614A proteins had normal primer extension capacity under the tested conditions (FIG.5A). However, whereas H614A was able to synthetize new primers with a similar pattern as the wild type piPolB (FIG.5B, lanes 6-7), K613A priming capacity was strongly reduced (lanes 4-5), suggesting a specific role of this residue during the de novo DNA synthesis.

It was next analyzed the TLS capacity of K613A variant by primer extension assay on the THF-containing template.FIG.5Cshows that the activity of K613A protein was strongly impaired compared to the wild type piPolB (lanes 3-4 vs. 7-8). Thus, although DNA primer synthesis and primer extension opposite to the undamaged and damaged substrates appears to rely on the same conserved catalytic residues, as shown for the D368A variant (see above), we were able to partially uncouple these activities. This result further confirms the unique intrinsic TLS and DNA primase capacities of piPolB and also unveils the role of the extended primer-stabilization motif of piPolB group that would be required for these activities.

Example 8. Biological Role of piPolB in De Novo DNA Synthesis

Considering the DNA priming capacity of piPolB, which is unprecedented in PolB family enzymes, it was decided to investigate its biological role in vivo. To this end, the piPolB-expressing bacteria were challenged with Mitomycin C (MMC) and ultraviolet-light (UV) irradiation, the two DNA damaging agents known to block DNA replication by introducing bulky base modifications and interstrand crosslinks (ICLs). Since piPolB was unable to replicate a T:T containing template (FIG.2D), it is unlikely that its TLS capacity may allow bypass of DNA damage induced by MMC treatment or UV-irradiation. However, given that replication blockage on the leading strand can be circumvented by re-priming events downstream of the UV-generated lesions, it was hypothesized that the de novo DNA synthesis by piPolB might contribute to relieving the genotoxic stress generated by DNA damaging agents. The results suggest that this is indeed the case, because expression of the wild type piPolB inE. coliB121(DE3) cultures significantly enhanced cell survival upon both MMC treatment and UV-irradiation, as compared with bacteria expressing D368A inactive piPolB variant (FIG.6). These results indicate a possible role of piPolB in DNA damage tolerance or repair in the context ofE. colicells.

Thus, the present invention reports the discovery and biochemical characterization of a new, previously overlooked major group of replicative PolBs, which were named herein “piPolBs” due to their unique capacity to perform primer-independent, templated DNA synthesis. Within the global PolB phylogeny, piPolB form a distinct, ancient clade on a par with the two previously described groups, rPolB and pPolB. The piPolB-encoding genes are found in MGEs, dubbed pipolins, most of which are integrated into genomes of bacteria from three different phyla (Firmicutes, Actinobacteria and Proteobacteria), but also replicating as circular plasmids in mitochondria. The distribution of pipolins is rather patchy, which is typical of integrated MGEs. To a large extent, pipolins seem to have coevolved with their hosts, because piPolB-based phylogeny is congruent with the general bacterial taxonomy (e.g., proteobacteria group together and are further divided into clades corresponding to different proteobacterial classes). Notably, phylogenetic analysis showed that piPolBs from mitochondrial plasmids cluster with proteobacterial homologs, in particular with those from alphaproteobacteria. Given that in all likihood mitochondria have evolved from an alphaproteobacterial ancestor at the onset of eukaryogenesis, it is tempting to speculate that piPolBs were introduced into eukaryotes along with the proto-mitochondrial alphaproteobacterial endosymbiont. According to conservative estimates based on the microfossil record, eukaryotes emerged ˜2 billion years ago. Thus, piPolB clade should be at least as old if not older, especially if the emergence of pipolins predated the divergence of the major bacterial phyla. However, the possibility that pipolins were horizontally introduced into mitochondria from proteobacteria in a more recent past cannot be excluded.

The piPolBs share the conserved active site with other PolBs and also contain TPR1 and TPR2 subdomains, a hallmark of pPolBs. Consistently, it was showed herein that piPolB displays efficient DNA polymerization and strand displacement activities. A more detailed biochemical characterization of piPolB also showed intrinsic TLS capacity across non-bulky base damages (FIG.2), which, although leading to mutation accumulation, will ultimately favor the maintenance of the damaged genome. Strikingly, unlike all other PolBs, piPolB does not require an externally provided primer for DNA replication. Conversely, it was found here that piPolB is able to initiate DNA synthesis de novo, a capacity so far exclusive to DNA primases. In the case of CD29DNAP, the TPR1 motif has been shown to make contacts with the template strand and to play a key role in the interaction with the TP during the early steps of protein-primed replication. Given that piPolBs in all likelihood do not interact with a TP, the function of TPR1 region may be limited to the interaction with the DNA. One additional possibility might be that this subdomain also interacts with certain cellular cofactors, which would modulate the piPolB activity in vivo. It was also noted that certain components of the cellular replication machinery (e.g., DNA ligase) might be involved in the pipolins' replication cycle.

The use of manganese as divalent cofactor instead of magnesium increased TLS across abasic sites (FIG.2C) as well as de novo DNA synthesis (FIGS.3C,9and4).

The enzyme tested in these examples acts both as a primase and a DNA polymerase. Here, it has been shown that piPolBs have a unique KTRG motif (SEQ ID NO: 46), alternative to the conserved KxY motif of PolBs, which interacts with the primer terminus. Moreover, an invariant lysine nearby the KTRG motif plays a key role both in TLS and de novo primer synthesis (FIG.5). Given the positive charge of this and nearby residues in the piPolB group, it is likely that the extended KX2-X3-10-KTRG motif (SEQ ID NO: 1) may induce a highly stable primer terminus binding mechanism that may favor the binding of the incoming nucleotide and the subsequent stabilization of the ternary complex, which would result in enhanced polymerization capacity. These results establish a structural liaison between TLS and priming capacities of piPolB.

As mentioned above, all DNA primases lack proofreading capacity. This seems advantageous for the efficient synthesis of short-lived Okazaki fragments. Conversely, the 3′-5′ exonuclease proofreading activity, which is necessary for faithful DNA replication by a DNA polymerase, could hinder the primase capacity. Thus, piPolB synthetic and degradative activities must be highly coordinated to allow efficient primer synthesis and faithful DNA replication. Furthermore, the piPolB exonuclease activity is also compatible with translesion synthesis of non-bulky base damages which, as reported previously for pPolB of bacteriophage Bam35, does not require template strand misalignment but tolerates damage-containing mismatches during processive DNA synthesis. Previous studies have shown that replication of pCRY1-like pipolins from fungal mitochondria can be initiated from multiple origins rather than from a fixed origin. However, this observation remained unexplained. These plasmids do not code for a putative TP; indeed, they only contain the piPolB gene. Thus, in the light of the results presented herein, such replication pattern is consistent with the possibility that pCRY1-like pipolins are replicated by their cognate piPolBs in a primer-independent manner. Analogously, the circular episomal form of bacterial pipolins could be replicated by piPolBs from multiple origins.

Replication across bulkier DNA lesions that could not be bypassed by piPolB might benefit from possible downstream re-priming. Accordingly, it has been shown here that expression of the wild type piPolB promotes survival ofE. colicells exposed to replication-blocking DNA damage agents (FIG.6). Hence, it was hypothesized that piPolB might have evolved to maintain pipolins' DNA by providing faithful and processive de novo DNA replication as well as tolerance to DNA damage, which may also increase the fitness of the host bacteria.

Importantly, piPolB holds a great promise for developing novel biotechnological applications. For instance, in vitro activities of piPolB, namely strand displacement and faithful, processive DNA polymerization, can be harnessed for efficient primer-independent whole genome amplification, whereas the translesion synthesis can be useful for amplification of damaged or ancient DNA templates. Given that piPolBs do not display strong sequence requirement for replication initiation, replication origins may be selected in a random manner, a property useful for whole genome amplification. piPolB could become a single-enzyme solution to achieve the goal of whole genome amplification in single-cell genomics applications.

Example 9. Amplification of Single- and Double-Stranded DNA by piPolB in the Absence of External Primers

To further analyze the application of piPolB for amplification of DNA without the addition of external primers, an optimization of the reaction conditions was first carried out using increasing concentrations of MgCl2as divalent cofactor, different reaction incubation times and M13 ssDNA input amount (FIG.12).

As shown inFIG.12A, the reaction is dependent of the metal concentration, with an optimum concentration of magnesium chloride of 15-30 mM. Thus, a concentration of 20 mM MgCl2was selected for subsequent experiments.

Amplified DNA can be detected after 5 h of reaction, although it increases with longer incubation time. In agreement with previous results, reaction product appeared as a smear of amplified DNA of a wide range of size lengths. It was then performed amplification of M13 ssDNA and dsDNA forms, using limiting DNA input amount (FIG.12B). Amplification of ssDNA could be detected with 1 ng DNA input (lane 4). Increasing DNA amount (lanes 5-8) gave rise to shorter DNA fragments, indicating distributive synthesis. Double-stranded DNA could be also amplified without any denaturing step, although it required higher amount of DNA input (lanes 14-15). Importantly, the reaction is highly specific, as no product is detected in the absence of DNA input (lanes 1-2,FIG.12Aand lanes 2 and 9,FIG.12B) or without metal (lanes 5-6,FIG.12A).

Samples inFIG.12Bwere digested with EcoRI, a single-cut reaction enzyme, prior to electrophoretic analysis. However, the smeared pattern is the same as inFIG.12A, indicating that the piPolB-generated DNA product would be mainly ssDNA. The use of ssDNA libraries has been successfully employed for amplification and subsequent sequencing of highly damaged ancient DNA. Furthermore, single-stranded library preparation can provide a higher yield and resolution level of DNA sequences from cell-free circulating DNA from blood and urine as well as DNA sequences from formalin-fixed, paraffin-embedded (FFPE) tissues. This, as well as the TLS of piPolB, suggest the potential application of primer-free amplification with piPolB for analysis of ancient and/or damaged DNA from a wide variety of sources.

As shown above, the reduced processivity of piPolB as compared with Φ29DNAP and other DNAPs, gives rise to a DNA amplified product that constitutes a DNA library of a wide range of length. Thus, we wondered if the joint use of piPolB and Φ29DNAP in the same reaction mixture would be able to produce a more homogeneous amplified product. As shown inFIG.13A, using both ssDNA and dsDNA, the addition of low piPolB concentration (12.5 nM, lanes 4 and 12) to a fixed concentration of Φ29DNAP is enough to produce large DNA fragments that can be resolved in dsDNA monomers of unit length. Furthermore, as shown inFIG.13B, the combined addition of piPolB and Φ29DNAP reduced the minimal input DNA required to 0.5 ng or below with either ssDNA and dsDNA.

Altogether, these results confirm the successful amplification of ssDNA and dsDNA, using piPolB in the absence of external primers, either by itself or together with another suitable DNAP, such as Φ29DNAP, and suggest multiple possible applications.

Example 10. Experimental Procedures

10.1. Bioinformatic Analyses.

Phylogenetic analysis. The non-redundant database of protein sequences at the NCBI was searched using the PSI-BLAST. For phylogenetic analyses protein sequences were aligned with the multiple sequence and structure alignment server PROMALS3D. Poorly aligned (low information content) positions were removed using the Gappyout function of Trimal. The dataset of viral, plasmid and polintons pDNAP sequences was collected previously (Krupovic, M., and Koonin, E. V., 2015, Nat Rev Microbiol, 13, 105-115). Maximum likelihood phylogenetic tree was constructed using the PhyML program the latest version of which includes automatic selection of the best-fit substitution model for a given alignment. The best model identified by PhyML was LG+G6+1+F (LG, Le-Gascuel matrix; G6, Gamma shape parameter: fixed, number of categories: 6; I, proportion of invariable sites: fixed; F, equilibrium frequencies: empirical).

Identification and annotation of integrated MGE. The pipolins' were identified thorough analysis of genomic neighborhoods of the piPolB-encoding genes. The precise boarders of integration were defined based on the presence of direct repeats corresponding to attachment sites. The repeats were searched for using Unipro UGENE. Pipolin genes were annotated based on the PSI-BLAST searches against the non-redundant protein database at NCBI and HHpred searches. Pipolins were compared to each other and visualized using the Genome Comparison Visualizer EASYFIG.

10.2. Protein Expression and Purification.

Primer-independent DNA polymerase (piPolB) fromE. coli3-373-03_S1_C2 Pipolin (NCBI GI:693097161, SEQ ID NO: 2) was obtained from GeneScript into NdeI-XhoI sites of pET23a. A stop codon was included to obtain the untagged recombinant protein (SEQ ID NO: 45). Polymerase (D368A) and exonuclease (D59A/E61A) deficient proteins, as well as wild type, K613A, H614A, K623A and R625A his-tagged variants, were obtained by site directed mutagenesis (Table 1):

TABLE 1Gene Sequence Information and mutagenesis primers.Oligonucleotide pairs used for site-directedmutagenesispiPolBvariantSequence (5′-3′)D368ACCCCGTCAGATCACTGGTATGATTACGCCCTGGCAGGCGCTTATACCACG (SEQ ID NO: 47)GCTGGTATAAGCGCCTGCCAGGGCGTAATCATACCAGTGATCTGACGGGG (SEQ ID NO: 48)D59A/CCCTGCATATCGGTTTTGCCACGGCATACGTGTTCAACCC61AGGAAACCC (SEQ ID NO: 49)GGGTTTCCGGGTTGAACACGTATGCCGTGGCAAAACCGATATGCAGGG (SEQ ID NO: 50)WTHisCCTTTTGCCTGCCGGTTTTACTCGAGCACCACCACCACCACCAC (SEQ ID NO: 51)GTGGTGGTGGTGGTGGTGCTCGAGTAAAACCGGCAGGCAAAAGG (SEQ ID NO: 52)K613AGGGTTCATCGATGCTGACCTGTGCACATGAAGTCTCTCAACTGATCGC (SEQ ID NO: 53)GCGATCAGTTGAGAGACTTCATGTGCACAGGTCAGCATCGATGAACCC (SEQ ID NO: 54)H614AGGGTTCATCGATGCTGACCTGTAAAGCTGAAGTCTCTCAACTGATCGC (SEQ ID NO: 55)GCGATCAGTTGAGAGACTTCAGCTTTACAGGTCAGCATCGATGAACCC (SEQ ID NO: 56)K623AGTCTCTCAACTGATCGCCATGGCAACCCGTGGTCAGCTGACG (SEQ ID NO: 57)CGTCAGCTGACCACGGGTTGCCATGGCGATCAGTTGAGAGAC (SEQ ID NO: 58)R625AGTCTCTCAACTGATCGCCATGAAAACCGCTGGTCAGCTGACGTATAAAGC (SEQ ID NO: 59)GCTTTATACGTCAGCTGACCAGCGGTTTTCATGGCGATCAGTTGAGAGAC (SEQ ID NO: 60)

Table 1. Gene Sequence Information and Mutagenesis Primers

All piPolB variants were expressed in B121(DE3) E. co/i cells, using ZYM-5052 autoinduction medium in the presence of 100 mg/L ampicillin. Cultures were grown for 20 h at 28° C. For purification of untagged piPolB variants, cells were disrupted by grinding with alumina and suspended in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 7 mM β-mercaptoethanol, 5% (v/v) glycerol) containing 1 M NaCl. Alumina and cell debris were removed by centrifugation, and absorbance at 260 nm was adjusted to 120 units/ml prior to DNA precipitation with 0.3% (w/v) polyethyleneimine. After centrifugation at 20,000×g for 20 min, ammonium sulfate was added to the supernatant to 69% saturation and centrifuged at 20,000×g for 30 min. The piPolB (wild type and mutants) containing pellet was resuspended in buffer A and applied to serial Q SEPHAROSE® FAST FLOW (GE Healthcare) anion exchange column and phosphocellulose (P11, Whatman) columns, at an ionic strength about 0.2 M NaCl. After extensive wash with increasing concentrations of NaCl in buffer A, purified DNA polymerase was eluted with 0.35 M NaCl and applied to Heparin-Sepharose® CL-6B affinity column (GE Healthcare), where, after washing with 0.35, 0.4 and 0.45 M NaCl, they were eluted at 1 M NaCl in buffer A.

Histidine-tagged variants were purified by standard method. Briefly, cells were resuspended in buffer C (50 mM phosphate buffer, pH 8, 7 mM β-mercaptoethanol, 5% (v/v) glycerol, 1 M NaCl, 5 mM imidazole) and incubated for 30 min at room temperature with 1 mg/mL lysozyme (Sigma) and 1 unit of benzonase (Sigma), prior to cell disruption by sonication. After centrifugation at 20,000×g for 30 min, the soluble fraction was applied to a Ni-NTA column (Qiagen). After extensive wash with 5, 10, 25 and 50 mM imidazole, the protein was eluted with 200 mM imidazole and subsequently applied to Heparin-Sepharose® CL-6B affinity column (GE Healthcare), where, after washing with 0.35, 0.4 and 0.45 M NaCl, it was eluted at 1 M NaCl in buffer A.

In all cases, pooled fractions containing pure piPolB variants were dyalized overnight against 500 volumes of buffer B (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 7 mM β-mercaptoethanol, 0.25 M NaCl and 50% (v/v) glycerol) and kept at −20° C., or at −70° C. for long storage. Final purity of the proteins was estimated to be >90% by SDS-PAGE followed by Coomassie blue staining.

10.3. Primer Extension Assays.

Oligonucleotides (Table 2) were purchased from Sigma in PAGE purification grade. To form a primer/template substrate as indicated in the top of each figure, the P15 oligonucleotide (Table 2) was 5′-labeled with [γ-32P]ATP using T4 Polynucleotide Kinase and hybridized to 1.2-fold molar excess of complementary unlabeled template oligonucleotides (T33GTA, T33GTT or T33GFA, Table 2) in the presence of 50 mM NaCl and 50 mM Tris-HCl, pH 7.5.

TABLE 2Sequences of oligonucleotides used in theexamples.NameSequence (5′-3′)P4GATC (SEQ ID NO: 61)P10GACTGCTTAC (SEQ ID NO: 62)P15GATCACAGTGAGTAC (SEQ ID NO: 63)T33GTAACTGGCCGTCGTTCTATTGTACTCACTGTGATC(SEQ ID NO: 64)T33GTTACTGGCCGTCGTTCTAATGTACTCACTGTGATC(SEQ ID NO: 65)T33GFTACTGGCCGTCGTTCTATFGTACTCACTGTGATC(SEQ ID NO: 66)P20-33GAACGACGGCCAGT (SEQ ID NO: 67)33AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO: 68)33T*TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT/invT/(SEQ ID NO: 69)CC31T*CCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT/invT/(SEQ ID NO: 70)15TCC16T*TTTTTTTTTTTTTTCCTTTTTTTTTTTTTTTTT/invT/(SEQ ID NO: 71)15TC17T*TTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTT/invT/(SEQ ID NO: 72)15TA17T*TTTTTTTTTTTTTTATTTTTTTTTTTTTTTTTT/invT/(SEQ ID NO: 73)M13 UPGTAAAACGACGGCCAGT (SEQ ID NO: 74)F represents the THF abasic site analog. */invT/ stands for a last dTMP nucleotide linked by an inverted 3′-3′ bond.

Assays were performed in 20 μL final volume containing 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 4% (v/v) glycerol, 0.1 mg/ml BSA, 0.05% (v/v) polyethylene glycol sorbitan monolaurate (TWEEN® 20) and, unless otherwise stated, 1 nM of the indicated 5′-labeled primer/template duplex, 10 nM DNA polymerase and the indicated dNTPs concentration. Reactions were triggered by addition of either 10 mM MgCl2or 1 mM MnCl2, as indicated and, after incubation for the indicated times at 30° C., the reactions were stopped by adding 10 μL of formamide loading buffer (98% formamide, 20 mM EDTA, 0.5% (w/v) bromophenol blue, and 0.5% (w/v) xylene cyanol). Samples were analyzed by 8 M urea-20% polyacrylamide gel electrophoresis (20×30×0.5 mm) in 1×TBE buffer. Gel bands were detected by an image analyzer either by autoradiography or phosphorimages (TYPHOON™ FLA 7000) and processed with IMAGE J SOFTWARE.

10.4. Replication of Single-Stranded DNA.

Genomic M13mp18 single-stranded circular DNA (laboratory stock) was diluted up to 50 ng/μL in a buffer containing 0.2 M NaCl and 60 mM Tris-HCl, pH 7.5 with or without M13 UP primer (Table 2), heated for 5 min at 65° C. and cooled slowly overnight to allow the annealing of the primer. Primed and non-primed M13 substrates were stored at −20° C. in small aliquots to minimize DNA nicking due to repetitive cycles of freeze-thaw.

The reaction mixture contained, in a final volume of 25 μL, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 4% (v/v) glycerol, 0.1 mg/ml BSA, 0.05% (w/v) polyethylene glycol sorbitan monolaurate (TWEEN® 20), 20 mM ammonium sulfate, 100 μM dNTPs, 0.5 μCi [α-32P]dATP, 3.2 nM of primed or non-primed M13mp18 ssDNA, and the indicated concentrations of each DNA polymerase. Reactions were triggered by addition of either 10 mM MgCl2or 1 mM MnCl2and incubated for 20 min at 30° C. Reactions were then quenched by adding 5 μL of 250 mM EDTA, 5% (w/v) SDS and directly loaded in TAE1× non-denaturing agarose electrophoresis. For alkaline agarose electrophoresis, an aliquot (15 μL) was subjected to gel filtration through Sephadex® G-15 gel filtration spin columns containing 0.1% (w/v) SDS. Lambda DNA ladder used as a size marker was labeled by filling-in with Klenow fragment (New England Biolabs) in the presence of [α-32P]dATP.

Replication of homopolymeric single stranded template was performed in similar conditions, using the 33-mer oligonucleotides (IDT) with the indicated sequences (Table 2), containing a terminal 3′-3′ inverted 3′-3′ inverted link to prevent both primer extension and exonucleolytic degradation. Oligonucleotide replication assays were performed in 20 μL final volume containing 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 4% (v/v) glycerol, 0.1 mg/ml BSA, 0.05% (v/v) polyethylene glycol sorbitan monolaurate (TWEEN® 20), 1 μM oligonucleotide template, 100 μM dNTPs, 500 nM of the indicated piPolB variant and 0.5 μCi [α-32P]dATP. After incubation for the indicated times at 30° C., the reactions were stopped by adding 10 μL of formamide loading buffer (98% formamide, 20 mM EDTA, 0.5% (w/v) bromophenol blue, and 0.5% (w/v) xylene cyanol). Samples were analyzed by 8 M urea-20% polyacrylamide analyzer either by autoradiography or phosphorimages (TYPHOON™ FLA 7000) and processed with IMAGE J SOFTWARE.

10.5. De Novo Primer Synthesis Detection.

To detect de novo primer synthesis [γ-32P]ATP was used as the labeled nucleotide. M13 ssDNA (3.2 nM) was used as template. The reaction mixture contained, in a final volume of 25 L, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 4% (v/v) glycerol, 0.1 mg/ml BSA, 0.05% (v/v) polyethylene glycol sorbitan monolaurate (TWEEN® 20), 10 μM dNTPs, 0.5 μCi [γ-32P]ATP, the indicated template and DNA polymerase (500 nM). Reactions were triggered by addition of either 10 mM MgCl2or 1 mM MnCl2and incubated for indicated times at 30° C. Then, the reactions were stopped by adding 10 μL of formamide loading buffer (98% formamide, 20 mM EDTA, 0.5% (w/v) bromophenol blue, and 0.5% (w/v) xylene cyanol). Samples were analyzed by 8 M urea-20% polyacrylamide gel electrophoresis (20×30×0.5 mm) in 1×TBE buffer. When indicated, high resolution gels (40 cm long) were used. Gel bands were detected by an image analyzer by phosphorimages (TYPHOON™ FLA 7000) and processed with IMAGE J SOFTWARE. The [γ32P]ATP-(dGMP)n DNA ladder used as size marker, generated by human PrimPol, was a gift from Dr. Luis Blanco (CBMSO, Madrid).

10.6. DNA Amplification Assays.

The reaction mixture contained, in a final volume of 10 μL, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 4% (v/v) glycerol, 0.1 mg/ml BSA, 0.05% (w/v) polyethylene glycol sorbitan monolaurate (TWEEN® 20), 10 mM ammonium sulfate, 500 μM dNTPs and, except otherwise stated, 20 mM MgCl2and 20 ng of either M13mp18 ssDNA or the counterpart dsDNA replicative form (RFI, New England Biolabs). Reactions were triggered by addition of the indicated DNA polymerase and incubated for the indicated times at 30° C., followed by incubation at 65° C. for 10 minutes and stored at 4° C. until analysis. When indicated, an aliquot (2 μL) was withdrawn and digested with EcoRI (EcoRI-HF, New England Biolabs), under standard conditions prior to analysis by TAE1× non-denaturing 0.8% agarose gel electrophoresis. DNA bands were visualized by subsequent staining with ethidium bromide (0.2 μg/mL). NZYDNA Ladder III molecular weight marker (NZYTech) were loaded for reference at each side of the gel.

10.7. Survival of piPolB Expressing Bacteria Upon DNA Damaging Agents Challenges.

Starter cultures ofE. coliB121(DE3) harboring pET23a::piPolB or pET23a::piPolB(D368A) plasmids were inoculated in LB media in the presence of ampicillin (150 μg/mL) and glucose (40 mM) and grown overnight shaking at 37° C. Saturated cultures were diluted (1:100) in fresh LB media with ampicillin and grown 1-2 h at 28° C. until DO600 nm=0.4. Recombinant protein expression was then induced by 0.5 mM IPTG during one hour prior to genotoxic challenge. At this point an aliquot was withdrawn to verify recombinant protein expression by SDS-PAGE (not shown). For MMC treatment, the indicated drug concentration was added directly into the cultures that were grown for an additional hour and then serial-diluted in fresh LB and plate onto LB-agar plates (without antibiotic). In the case of UV-exposure, the induced cultures were serial-diluted in sterile PBS and plated onto LB-agar plates prior to irradiation with the indicated UV-light intensity in a spectroline UV crosslinker SPECTROLINKER™ XL-1000, Spectronics Corporation).

Data analysis and representation was performed using R and R-Studio (Studio, Inc., Boston, MA), using packages Dplyr, Stats and Ggplot2, available from CRAN (The comprehensive R archive network). Based on Shapiro-Wilk normality tests, results from MMC and UV light challenges were analyzed by Paired T-test and Dependent 2-group Wilcoxon Signed Rank Test, respectively. P-values are indicated in theFIG.6as *p≤0.1, **p≤0.05 and ***p≤0.01.

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