Here we sought to analyze whether rNMP incorporation into

Here, we sought to analyze whether rNMP incorporation into DNA is a conserved property of DNA synthesis in Archaea. Up to now, four DNA Pol families, (B, D, X and Y) and the p41/p46 primase–polymerase have been found in the genomes of 251 archaeal species (https://gold.jgi.doe.gov/). In vitro activities of families B, D and Y and the p41/p46 complex of archaeal DNA Pols have been demonstrated, but never characterized for the X-family. Which DNA Pols are responsible for duplicating the archaeal genome is not currently known with the same degree of certainty as for Bacteria or Eukarya. All I-BET-762 mg contain a family-B DNA Pol (PolB), usually present as several members in the Crenarchaea and as a single enzyme in the Euryarchaea. With the exception of the Crenarchaea, a HiFi family-D DNA Pol (PolD) is found in all Archaea and is unique to this domain. Archaea also possess a p41/p46 primase–polymerase complex with p41 belonging to archaeo-eukaryotic primase (AEP) family. At the replication fork in the Crenarchaea, it is believed that the p41/p46 complex initiates DNA replication by synthesizing an RNA primer, followed by leading and lagging strand DNA synthesis using HiFi family-B DNA Pols [27], [28], [29]. While it is clear that p41/p46 is involved in the initiation process in the Euryarchaea [30], [31], [32], the role of HiFi PolB and/or PolD acting selectively on opposite DNA strands at the replication fork remains inconclusive because of biochemical and genetic divergences [33], [34], [35], [36], [37], [38], [39]. Similar to the DNA repair family-Y DNA Pol [28], [40], [41], it is not excluded that the p41/p46 complex, like families B and D DNA Pols, might operate in other archaeal DNA transactions (repair, damage tolerance, damage signaling, etc.) [14], [27], [31], [33], [42], [43], [44], [45], [46]. In the present work, we ask whether rNMP incorporation is an evolutionarily conserved property of DNA synthesis in Archaea. For this purpose, we used the best characterized hyperthermophilic anaerobe, Pyrococcus abyssi GE5 [47]. This strain duplicates its genome as fast as Bacteria, supported by eukaryotic-like replication proteins [48]. Roles have been proposed whereby RNA priming is performed by the p41/p46 complex [31], followed by leading and lagging strand DNA synthesis by PolB and PolD, respectively [35], [49], [50],. Maturation of Okazaki fragments likely involves PolD but precludes any contribution of PolB in the absence of RNase HII [34]. Beyond these crucial roles in DNA replication, all three DNA polymerizing enzymes seem to work in DNA transactions such as repair, damage signaling or tolerance [31], [42], [43], [51]. We further analyzed whether rNMP incorporation by PolB, PolD and the p41/p46 complex is conserved during DNA synthesis. At the physiological dNTP and rNTP levels, we demonstrate that PolD is the main DNA Pol able to insert rNMPs in an error-free manner. In addition, we report that single embedded rNMPs are bypassed by all three DNA Pols with variable nucleotide incorporation/misincorporation proficiency regardless of the nature of the base and sequence context. Unexpectedly, we also discovered that PolD can incorporate a single rNMP opposite template ribonucleotides. These results suggest that rNMP incorporation into DNA represents a conserved feature of archaeal DNA Pols, which may have various consequences for their genome integrity.