Introduction Maintenance of genome integrity is essential fo
Introduction Maintenance of genome integrity is essential for life, and faithful DNA replication and repair ensure this. One of the most important parameters for DNA maintenance is the regulation of optimal concentrations of the four 5′-deoxyribonucleoside-triphosphates (dNTPs) the precursors for DNA synthesis, by ribonucleotide reductase (RNR) . RNR, which reduces the 5′-ribonucleoside-triphosphates (rNTPs) to the corresponding dNTPs, also maintains the ratio of rNTPs to dNTPs at distinct levels throughout the cell cycle. Moreover, the relative abundance of nucleotides is dependent on the type of tissue, organelle and cell, with rNTP content significantly exceeding dNTP content , , . For these reasons, high- and low-fidelity DNA polymerases (DNA Pols) face the challenge of selecting the correct dNTPs over the more abundant rNTPs during the normal processes of DNA replication and repair. While cellular concentrations of these nucleotides are documented in Eukarya and Bacteria, they remain largely unknown in Wnt-C59 . Other important parameters such as DNA Pol selectivity, proofreading 3′ → 5′ exonuclease activity and mismatch repair (MMR) contribute to improving the fidelity of DNA synthesis , . With regard to rNMP incorporation, high- and low-fidelity DNA Pols have evolved structural features for blocking rNTP entry into their active sites. A steric gate residue or a protein backbone segment is believed to generate a clash with the 2′-hydroxyl group of the ribose of the incoming rNTP . This prevention mechanism is of particular importance for the proofreading function of high-fidelity (HiFi) DNA Pols since it is more proficient in the excision of incorrect dNMPs than rNMPs from extending primers , . Recent data indicate that rNMPs are the main non-canonical nucleotides incorporated into DNA  by eukaryotic and bacterial DNA Pols, with surprisingly high frequency , . rNMP incorporation may also arise from imperfect processing of Okazaki fragments . The biological evidence of embedded rNMPs in the DNA of Bacteria, Archaea and Eukarya is based on in vivo studies showing an increased load of genomic rNMPs and genetic instability in ribonuclease H (RNase H)-defective cells , , , , . Because of the reactive 2′-hydroxyl group on the ribose ring, rNMPs in the chromosome make the DNA strand susceptible to spontaneous and enzymatic hydrolytic cleavage. They can also modify the helical structure in DNA that possibly interferes with cellular DNA transactions . To prevent persistent rNMP accumulation in genomic DNA, cells evolved a specific repair pathway termed ribonucleotide excision repair (RER), in which the principal enzyme is type 2 RNase H (RNase H2 or HII) , , . In the absence of RNase H2, a topoisomerase 1 (Top1 or topA gene) repair pathway can remove some rNMPs in yeast , . Moreover, rNMPs might also be targeted by other mechanisms such as MisMatch Repair (MMR), nucleotide excision repair (NER) and base excision repair (BER) , , , . 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 Archaea 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 , , . While it is clear that p41/p46 is involved in the initiation process in the Euryarchaea , , , 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 , , , , , , . Similar to the DNA repair family-Y DNA Pol , , , 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.) , , , , , , , , .