Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh

Abstract

Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh 2 strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous 21 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.KEYWORDS trans-lesion DNA synthesis (TLS); abasic site; 7,8-dihydro-8-oxo-deoxyguanosine; oligonucleotide-mediated transformation T HE fact that DNA damage occurs in all living cells poses a threat to the accurate replication and partitioning of their genomes. Cells counter this threat with an array of diverse damage-coping systems, some of which repair the DNA, whereas others enable replication to continue past persistent lesions. Lesion bypass, also termed "damage tolerance," can follow various alternatives, which are broadly distinguished as error-free vs. error-prone (Lehmann et al. 2007). The error-free pathways generally use recombinationassociated functions to pair a strand, newly synthesized on intact template, to the damaged DNA strand; the mechanisms for this include transient reversal of the replication fork and strand exchange at gaps left behind the fork (Sale 2012). These mechanisms bypass lesions accurately, although they can promote other forms of genetic instability, such as ectopic recombination between dispersed repeats (Izhar et al. 2013).Error-prone mechanisms of lesion bypass, in contrast, use specialized, trans-lesion synthesis (TLS) polymerases to continue strand elongation using the damaged template; most of these enzymes belong to t...

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

“…As expected, the pol + recipient demonstrated the highest relative transformation efficiency (0.659), representing a limited impact of G oxidation on oligonucleotide incorporation and replication in normal cells. The corresponding transformation efficiency of the Δ dbh recipient strain was about 10% of this value (0.068), indicating that loss of the Y-family DNAP Dbh compromised the cell’s ability to tolerate oxidative damage, consistent with the biochemical and genetic properties of this DNAP ( Sakofsky et al, 2012 ; Sakofsky and Grogan, 2015 ; Jain et al, 2020 ). The triple polymerase mutant yielded a relative transformation efficiency similar to that of the Δdbh recipient.…”

Heterotrimeric PCNA increases the activity and fidelity of Dbh, a Y-family translesion DNA polymerase prone to creating single-base deletion mutations

“…Inactivation of the dbh gene in S. acidocaldarius does not change the overall spontaneous mutation rate, but the mutation spectrum changes significantly, with fewer insertion-deletion mutations and more base-substitution mutations [28]. Additional studies indicate that Dbh is primarily responsible for the accurate bypass of 8-oxo-dG lesions, by correctly inserting dC opposite the lesion up to 90% of the time, but does not increase the frequency of single base deletion mutations in a 5'-GY n repetitive sequence that would be a deletion hotspot in vitro [29].…”

Heterotrimeric PCNA Increases the Activity and Fidelity of Dbh, a Y-family Translesion DNA Polymerase Prone to Creating Single-Base Deletion Mutations

“…Inactivation of the dbh gene in S. acidocaldarius does not change the overall spontaneous mutation rate, but the mutation spectrum changes significantly, with fewer insertion-deletion mutations and more base-substitution mutations [28]. Additional studies indicate that Dbh is primarily responsible for the accurate bypass of 8-oxo-dG lesions, by correctly inserting dC opposite the lesion up to 90% of the time, but does not increase the frequency of single base deletion mutations in a 5'-GY n repetitive sequence that would be a deletion hotspot in vitro [29].These findings suggest that Dbh is specifically recruited to sites of oxidative DNA damage and, because its access to replicating DNA is restricted, it does not introduce deletion mutations into the genome to the extent that the biochemical data would indicate. However, the intrinsic fidelity of Dbh may also increase in the cellular environment.…”