To identify DNA-oxidation defenses of hyperthermophilic archaea, we deleted genes encoding the putative 7,8-dihydro-8-oxoguanine (oxoG)-targeted N-glycosylase of S. acidocaldarius (ogg; Saci_01367), the Y-family DNA polymerase (dbh; Saci_0554), or both, and measured the effects on cellular survival, replication accuracy, and oxoG bypass in vivo. Spontaneous G:C to T:A transversions were elevated in all Δogg and Δdbh constructs, and the Δogg Δdbh double mutant lost viability at a faster rate than isogenic WT and ogg strains. The distribution of G:C to T:A transversions within mutation-detector genes suggested that reactivity of G toward oxidation and the effect on translation contribute heavily to the pattern of mutations that are recovered. An impact of the Ogg protein on overall efficiency of bypassing oxoG in transforming DNA was evident only in the absence of Dbh, and Ogg status did not affect the accuracy of bypass. Dbh function, in contrast, dramatically influenced both the efficiency and accuracy of oxoG bypass. Thus, Ogg and Dbh were found to work independently to avoid mutagenesis by oxoG, and inactivating this simple but effective defense system by deleting both genes imposed a severe mutational burden on S. acidocaldarius cells. IMPORTANCE Hyperthermophilic archaea are expected to have effective (and perhaps atypical) mechanisms to limit the genetic consequences of DNA damage, but few gene products have been demonstrated to have genome-preserving functions in vivo. This study confirmed by genetic criteria that the S. acidocaldarius Ogg protein avoids the characteristic mutagenesis of G oxidation. This enzyme and the bypass polymerase Dbh have similar impacts on genome stability but work independently, and may comprise most of the DNA-oxidation defense of S. acidocaldarius. The critical dependence of accurate oxoG bypass on the accessory DNA polymerase Dbh further argues that some form of polymerase exchange is important for accurate genome replication in Sulfolobus, and perhaps in related hyperthermophilic archaea.

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The thermophilic crenarchaeon Sulfolobus acidocaldarius has four DNA polymerases (DNAPs): PolB1, PolB2, PolB3, and Dbh (PolY). Previous in vitro studies suggested that PolB1 is the main replicative DNAP of Sulfolobales whereas PolB2 and Y-family polymerases Dpo4 (Saccharolobus solfataricus) or Dbh are involved in DNA repair and translesion DNA synthesis. On the other hand, there are various opinions about the role of PolB3, which remains to be clearly resolved. In order to examine the roles of the DNAPs of S. acidocaldarius through in vivo experiments, we constructed polB2, polB3, and dbh deletion strains and characterized their phenotypes. Efforts to construct a polB1 deletion strain were not successful; in contrast, it was possible to isolate triple gene-deletion strains lacking polB2, polB3, and dbh. The growth of these strains was nearly the same as that of the parent strains under normal growth conditions. The polB2, polB3, and dbh single-deletion strains were sensitive to some types of DNA-damaging treatments, but exhibited normal sensitivity to UV irradiation and several other damaging treatments. Overall, the genotype which exhibited the greatest sensitivity to the DNA-damaging treatments we tested was the ΔpolB2 ΔpolB3 combination, providing the first evidence of overlapping function for these two DNAPs in vivo. The results of our study strongly suggest that PolB1 is responsible for the DNA replication of both the leading and lagging strands and is sufficient to complete the repair of most DNA damage under normal growth conditions in S. acidocaldarius.

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