Genetic Control of Oxidative Mutagenesis in Sulfolobus acidocaldarius

Abstract

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.

PolB1 Is Sufficient for DNA Replication and Repair Under Normal Growth Conditions in the Extremely Thermophilic Crenarchaeon Sulfolobus acidocaldarius

“…Previous in vivo, in vitro, and bioinformatic studies suggest that PolB1 is the main replicative DNAP in these archaea (Choi et al, 2011;Makarova et al, 2014). The biochemical evidence that PolB2 and Dbh may be specialized for DNA repair, TLS, or both (Boudsocq et al, 2004;Choi et al, 2011) has been supported by phenotypic analysis of dbh (polY) mutants of S. acidocaldarius (Sakofsky et al, 2012;Jain et al, 2020), and dpo2 (polB2) mutants of a related genus (Feng et al, 2020). Although the PolB3 class of polymerases has the widest distribution among archaea (Cooper, 2018), its functional significance remains unclear.…”

“…The purified PCR products (200 ng/µL in 5 mM Tris-HCl, pH 8.5) were used for subsequent electrotransformation. Production of targeted pyrE cassettes and replacement of the Saci_0074, Saci_0554, and Saci_2156 genes followed the scheme described previously for the Saci_0554 and Saci_1367 genes (Jain et al, 2020).…”

“…The identity of the nucleotide inserted opposite the oxoG in individual transformants was determined by restriction analysis. After clonal purification of each transformant, the pyrE gene was amplified and treated with restriction endonucleases that discriminate among the four possible bases inserted at the query site (Jain et al, 2020).…”

“…in the chromosomes of normal, dbh, and triple-deletion polymerase mutants of S. acidocaldarius. The assay selects for cells that have been genetically transformed by synthetic singlestranded oligonucleotides carrying 8oxoG at a synonymous position within the pyrE gene sequence (Jain et al, 2020). Thus, successful transformation requires incorporation of the oligonucleotide into the recipient chromosome and bypass of the lesion in the first round of replication, but any of the four nucleotides inserted opposite the lesion generates a transformant.…”

“…Thus, successful transformation requires incorporation of the oligonucleotide into the recipient chromosome and bypass of the lesion in the first round of replication, but any of the four nucleotides inserted opposite the lesion generates a transformant. At this position each nucleotide creates a distinct restriction site, however, allowing the inserted nucleotide to be identified by restriction assays of the PCR products (Jain et al, 2020).…”