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Primer-Independent DNA Synthes Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements Modesto Redrejo-Rodríguez, Carlos Ordonez, Mónica Berjón-Otero, Juan Moreno-González, Cristian Aparicio-Maldonado, Patrick Forterre, Margarita Salas, Mart Krupovic To cite this version: Modesto Redrejo-Rodríguez, Carlos Ordonez, Mónica Berjón-Otero, Juan Moreno-González, Cristian Aparicio-Maldonado, et al.. Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements. Cell Reports, Elsevier Inc, 2017, 21 (6), pp.1574- 1587. 10.1016/j.celrep.2017.10.039. pasteur-01977354 HAL Id: pasteur-01977354 https://hal-pasteur.archives-ouvertes.fr/pasteur-01977354 Submitted on 10 Jan 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial - ShareAlike| 4.0 International License Article Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements Graphical Abstract Authors Modesto Redrejo-Rodrı´guez, Carlos D. Ordo´ n˜ ez, Mo´ nica Berjo´ n-Otero, ..., Patrick Forterre, Margarita Salas, Mart Krupovic Correspondence [email protected] (M.R.-R.), [email protected] (M.S.), [email protected] (M.K.) In Brief Redrejo-Rodrı´guez et al. report and characterize a DNA polymerase group (piPolB) from the B family that can perform primer-independent DNA replication. PiPolBs are encoded by Pipolins, diverse self-replicating genetic elements that are widespread among bacterial phyla and in mitochondria. Highlights d Primer-independent PolBs (piPolBs) display templated de novo DNA synthesis capacity d piPolBs denote a third major group of family B DNA polymerases d piPolBs are the hallmark of pipolins, self-replicating mobile genetic elements d Pipolins are widespread among diverse bacterial phyla and mitochondria Redrejo-Rodrı´guez et al., 2017, Cell Reports 21, 1574–1587 November 7, 2017 ª 2017 The Authors. https://doi.org/10.1016/j.celrep.2017.10.039 Cell Reports Article Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements Modesto Redrejo-Rodrı´guez,1,5,* Carlos D. Ordo´ n˜ ez,1 Mo´ nica Berjo´ n-Otero,1,3 Juan Moreno-Gonza´ lez,1 Cristian Aparicio-Maldonado,1,4 Patrick Forterre,2 Margarita Salas,1,* and Mart Krupovic2,* 1Centro de Biologı´a Molecular ‘‘Severo Ochoa,’’ Consejo Superior de Investigaciones Cientı´ficas and Universidad Auto´ noma de Madrid, Universidad Auto´ noma, Cantoblanco, 28049 Madrid, Spain 2Institut Pasteur, Unite´ Biologie Mole´ culaire du Ge` ne chez les Extremophiles,^ Paris, France 3Present address: Max Plank Institute for Medical Research, Heidelberg, Germany 4Present address: Department of Bionanoscience, Delft University of Technology, Delft, the Netherlands 5Lead Contact *Correspondence: [email protected] (M.R.-R.), [email protected] (M.S.), [email protected] (M.K.) https://doi.org/10.1016/j.celrep.2017.10.039 SUMMARY 2002): RNA primed (rPolBs) and protein primed (pPolBs). The evolutionary relationship between the two groups is unknown, Family B DNA polymerases (PolBs) play a central role and it is thus unclear whether the ancestral PolB would have em- during replication of viral and cellular chromosomes. ployed a protein or an RNA oligonucleotide as a primer. The rPolB Here, we report the discovery of a third major group group contains mainly replicases devoted to accurate and of PolBs, which we denote primer-independent efficient copying of large cellular and viral genomes. By contrast, PolB (piPolB), that might be a link between the pre- pPolBs are exclusive to selfish mobile genetic elements (MGEs) viously known protein-primed and RNA/DNA-primed and viruses with moderately sized linear genomes (<50 kb) (Ka- zlauskas and Venclovas, 2011; Krupovic and Koonin, 2015). PolBs. PiPolBs are encoded by highly diverse pipolins The signature of pPolBs is the presence of specific subdomains, mobile genetic elements, , integrated in the named TPR1 and TPR2, which were originally described in F29 genomes of diverse bacteria and also present as cir- DNAP. TPR1 is required for the DNAP interaction with the TP, cular plasmids in mitochondria. Biochemical charac- whereas TPR2 endows pPolB with the processivity and strand terization showed that piPolB displays efficient DNA displacement capacity (Dufour et al., 2000; Rodrı´guez et al., polymerization activity that can use undamaged 2005; Salas et al., 2016), the two properties ensuring the superi- and damaged templates and is endowed with ority of F29DNAP in various molecular biology applications, such proofreading and strand displacement capacities. as multiple-displacement single-molecule DNA amplification Remarkably, the protein is also capable of tem- (Hutchison et al., 2005; Sidore et al., 2016). plate-dependent de novo DNA synthesis, i.e., DNA- Protein priming has been described for a number of viruses priming activity, thereby breaking the long-standing (Berjo´ n-Otero et al., 2016; Hoeben and Uil, 2013; Salas, 1991) as well as for linear eukaryotic plasmids (Klassen and Meinhardt, dogma that replicative DNA polymerases require a 2007). More recently, pPolB-encoding genes were also identified pre-existing primer for DNA synthesis. We suggest in two superfamilies of MGE integrated into various cellular that piPolBs are involved in self-replication of pipo- genomes (Krupovic and Koonin, 2016). The first superfamily lins and may also contribute to bacterial DNA dam- comprises eukaryotic virus-like transposable elements, called age tolerance. Polintons (also known as Mavericks), which, besides pPolB, encode retrovirus-like integrases and a set of proteins predicted INTRODUCTION to be involved in the formation of viral particles (Krupovic et al., 2014a). The second supergroup, denoted casposons, is present DNA polymerases (DNAPs) are key enzymes essential for in a wide range of archaea and some bacteria (Krupovic genome replication, recombination, and repair across all cellular et al., 2014b). For integration into the cellular genome, caspo- life forms and their viruses. Family B DNAPs (PolBs) are involved sons employ endonucleases homologous to Cas1, a signature in genome replication in Eukarya and Archaea, but also in viruses enzyme of the CRISPR-Cas systems (Be´ guin et al., 2016; Kru- from the three domains of life (Koonin, 2006). For the initiation of povic et al., 2017). It has been postulated that pPolBs participate DNA synthesis, all PolBs characterized thus far depend on the in the replication of the casposon and polinton genomes (Kapito- presence of an external primer, a hydroxyl group presented either nov and Jurka, 2006; Krupovic et al., 2014b); accordingly, these by a nucleic acid (RNA or DNA) or the so-called terminal protein MGEs are referred to as self-synthesizing or self-replicating (TP). Thus, based on the primer requirement for the initiation of elements. genome replication as well as phylogenetic clustering, these en- Here we uncover a highly diverse superfamily of self-repli- zymes can be broadly divided into two major groups (File´ e et al., cating MGEs, dubbed pipolins, which are present in three major 1574 Cell Reports 21, 1574–1587, November 7, 2017 ª 2017 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). bacterial phyla, as well as in mitochondria, and encode divergent two major groups, which are roughly congruent with the bacterial PolB carrying TPR1 and TPR2 subdomains. Biochemical char- taxonomy (Figure 1C; Figure S1). The first one predominantly in- acterization of a representative enzyme encoded by a pipolin cludes sequences from Actinobacteria and several orders of Fir- from Escherichia coli showed that the protein displays a versatile micutes, namely, Bacillales, Lactobacillales, and Clostridiales. and efficient DNA replication capacity. Strikingly, the protein is The second group contains sequences from different classes also capable of an intrinsic de novo DNA synthesis, i.e., DNA- of Proteobacteria. Notably, the latter group also includes piPolBs priming activity, not previously described in members of the from circular mitochondrial plasmids, which cluster with se- PolB family. This group of DNAPs, which we denote primer-inde- quences from alphaproteobacteria (Figure 1C; Figure S1). pendent PolB (piPolB), should be sufficient to initiate and carry out an entire replication cycle of the circular pipolin DNA in vivo. piPolBs Are Encoded within Self-Replicating MGEs Moreover, enhanced survival of E. coli cells expressing piPolB Genomic context analysis provided compelling evidence that the upon replication blockage by DNA-damaging agents suggests majority of piPolBs are encoded within MGEs integrated into bac- an additional role of piPolB in bacterial DNA damage tolerance. terial chromosomes. Unlike casposons and polintons, the vast majority of piPolB-carrying MGEs encoded integrases of the RESULTS tyrosine recombinase superfamily (Y-integrases). Some of the el-
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