Archaeal DNA Polymerase D but Not DNA Polymerase B Is Required for Genome Replication in Thermococcus kodakarensis

Abstract

c Three evolutionarily distinct families of replicative DNA polymerases, designated polymerase B (Pol B), Pol C, and Pol D, have been identified. Members of the Pol B family are present in all three domains of life, whereas Pol C exists only in Bacteria and Pol D exists only in Archaea. Pol B enzymes replicate eukaryotic chromosomal DNA, and as members of the Pol B family are present in all Archaea, it has been assumed that Pol B enzymes also replicate archaeal genomes. Here we report the construction of Thermococcus kodakarensis strains with mutations that delete or inactivate key functions of Pol B. T. kodakarensis strains lacking Pol B had no detectable loss in viability and no growth defects or changes in spontaneous mutation frequency but had increased sensitivity to UV irradiation. In contrast, we were unable to introduce mutations that inactivated either of the genes encoding the two subunits of Pol D. The results reported establish that Pol D is sufficient for viability and genome replication in T. kodakarensis and argue that Pol D rather than Pol B is likely the replicative DNA polymerase in this archaeon. The majority of Archaea contain Pol D, and, as discussed, if Pol D is the predominant replicative polymerase in Archaea, this profoundly impacts hypotheses for the origin(s), evolution, and distribution of the different DNA replication enzymes and systems now employed in the three domains of life. DNA replication, an essential event for all cellular life, is catalyzed by protein complexes designated replisomes, in which individual activities are tightly regulated and coordinated. DNA polymerases are the functional center of the replisome, but structurally distinct DNA polymerases, designated family C (Pol C) and family B (Pol B) polymerases, catalyze genome replication in Bacteria and eukaryotes, respectively (1-3). This difference has led to much debate, most fundamentally regarding whether DNA replication has evolved more than once, possibly independently in different biological lineages (1, 4-10). All known archaeal genomes encode at least one member of the Pol B family, and given that Archaea are evolutionarily closer to eukaryotes than are Bacteria (11, 12), it has been tacitly assumed, but challenged (13,14), that Pol B enzymes must also replicate archaeal genomes. Presumably, this must be the case for the Crenarchaeota, as their genomes appear to encode only Pol B enzymes. This is, however, only an assumption for all members of the Euryarchaeota, Thaumarchaeota, Korarchaeota, Aigarchaeota, and Nanoarchaeota lineages, as their genomes encode not only Pol B enzymes but also members of an archaeon-specific DNA polymerase family designated Pol D (Fig. 1) (11, 13-15).The Thermococcales are hyperthermophilic Euryarchaea, and given the commercial value of thermostable processive DNA polymerases, Pol B polymerases from this genus have received extensive characterization (13,16,17). Within these single polypeptide enzymes, the regions and residues directly responsible for nucleotide polymerization, 3=¡...

The apt /6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus

“…The profile of frameshifts, tandem duplications, and larger deletion and BPSs obtained in this work was generated from molecular analysis of the apt locus from over 200 6-MP r strains. We found that the mutation profile is comparable to that of T. kodakarensis, also assayed by 6-MP counterselection with an hpt gene (49). Interestingly, a 5-FOAbased forward mutation assay in S. acidocaldarius revealed that frameshift mutations are dominant in this organism (50).…”

“…Interestingly, a 5-FOAbased forward mutation assay in S. acidocaldarius revealed that frameshift mutations are dominant in this organism (50). Furthermore, this and a few other studies have revealed that the transitions outnumbered transversions by more than 2-fold, and a very strong strand bias for G or C-to-A or T transitions was observed (49,50,52,53). These should be further studied by generating mutants deficient in certain DNA repair mechanisms and investigating their effect on the profile of spontaneous mutations.…”

The archaeal DNA replication machinery: past, present and future

“…This would make archaeal organisms similar to eukaryotic organisms, in which Polε replicates the leading strand while Polδ replicates the lagging strand. Using gene knockouts, however, it was found that, at least in some species, PolB is not required for cell viability (Cubonova et al, 2013;Sarmiento et al, 2013). These results suggest that only PolD is the replicative enzyme in those species.…”

Archaeal DNA polymerases in biotechnology

“…A helical interdomain insertion is green. Other motifs are colored as follows: the uracil-binding motif is red, the "DIE" in the Exo I motif is green, residue D215 in the Exo II motif is white gray, residue D315 in the Exo III motif is yellow, the "YxGG" motif is white gray, the Motif A is green, the Motif B is blue and the Motif C is magenta for genome replication in T. kodakarensis (Cubonova et al 2013). Although biochemical data suggested that pol Ds from P. furiosus and P. abyssi were suitable enzymes for PCR (Killelea et al 2014), these DNA pols have not been commercialized so far.…”