Here we uncover a highly diverse superfamily of
Here we uncover a highly diverse superfamily of self-replicating MGEs, dubbed pipolins, which are present in three major bacterial phyla, as well as in mitochondria, and encode divergent PolB carrying TPR1 and TPR2 subdomains. Biochemical characterization of a representative enzyme encoded by a pipolin from Escherichia coli showed that the protein displays a versatile and efficient DNA replication capacity. Strikingly, the protein is also capable of an intrinsic de novo DNA synthesis, i.e., DNA-priming activity, not previously described in members of the PolB family. This group of DNAPs, which we denote primer-independent PolB (piPolB), should be sufficient to initiate and carry out an entire replication cycle of the circular pipolin DNA in vivo. Moreover, enhanced survival of E. coli pge2 expressing piPolB upon replication blockage by DNA-damaging agents suggests an additional role of piPolB in bacterial DNA damage tolerance.
Discussion Here we report the discovery and biochemical characterization of a previously overlooked major group of replicative PolBs, which we named piPolB due to their unique capacity to perform primer-independent, templated DNA synthesis. Within the global PolB phylogeny, piPolB forms a distinct, ancient clade on 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 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 (Forterre, 2012, Makarova et al., 2014). To a large extent, pipolins seem to have co-evolved with their hosts, because piPolB-based phylogeny is congruent with the general bacterial taxonomy. Notably, phylogenetic analysis showed that piPolBs from mitochondrial plasmids cluster with alphaproteobacterial homologs (Figure S1). Given that in all likelihood mitochondria have evolved from an alphaproteobacterial ancestor at the onset of eukaryogenesis (Gray, 2012), 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 (Dyall et al., 2004, López-García and Moreira, 2015). Thus, the 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. The piPolBs share the conserved active site as well as the TPR1 and TPR2 subdomains with pPolBs (Figure 1). Consistently, we showed that piPolB displays efficient DNA polymerization and strand displacement activities. Furthermore, piPolB also showed intrinsic TLS capacity across non-bulky base damages (Figure 3). Strikingly, unlike all other PolBs, piPolB does not require an externally provided primer for DNA replication. Conversely, we found that piPolB is able to initiate DNA synthesis de novo, a capacity so far exclusive to DNA primases. In the case of Φ29DNAP, the TPR1 motif makes contacts with the template strand and plays a key role in the interaction with the TP during the early steps of protein-primed replication (Dufour et al., 2000, Kamtekar et al., 2006). Given that piPolBs do not interact with a TP, the function of TPR1 region may be limited to the interaction with the DNA or certain cellular cofactors, which would modulate the piPolB activity in vivo. The use of manganese as divalent cofactor instead of magnesium increased TLS across abasic sites (Figure 3C) as well as de novo DNA synthesis (Figures 4C and 5; Figure S5). Although the roles of divalent metal ions in DNA synthesis have been controversial, a number of recent findings suggest a physiological role of manganese ions as a cofactor in DNA damage tolerance and repair pathways (Andrade et al., 2009, Cannavo and Cejka, 2014, Kent et al., 2016), as in the case of the human PrimPol (García-Gómez et al., 2013). PrimPol domain-containing DNA primases have been shown to possess multiple enzymatic activities in vitro, including primer-dependent and primer-independent DNAP activity, nucleotidyl-transferase, TLS, and even reverse-transcriptase activities (Gill et al., 2014, Guilliam et al., 2015, Iyer et al., 2005, Lipps et al., 2003, Martínez-Jiménez et al., 2015). Although in certain virus- or plasmid-encoded proteins the PrimPol domain is fused to various helicases and can synthesize large DNA products (Zhu et al., 2017), these enzymes lack the exonuclease domain, and their DNA polymerization on longer templates appears to be mainly distributive. Thus, it is generally considered that the role of PrimPol proteins in vivo is largely restricted to the synthesis of short primers, which are extended by the cellular replicative DNAPs (Beck et al., 2010, Gill et al., 2014). By contrast, piPolBs are full-fledged replicative DNAPs endowed with the proofreading and strand displacement capacities. Thus, our results challenge a long-standing dogma in the field, which states that replicative DNAPs are unable to synthesize DNA de novo, without a pre-existing primer providing a hydroxyl moiety to anchor the incoming nucleotide (Kornberg and Baker, 1992, Kuchta and Stengel, 2010).