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(-) 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 -1 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: 7,8-dihydro-8-oxo-deoxyguanosine; abasic site; oligonucleotide-mediated transformation; trans-lesion DNA synthesis (TLS).

Figure 1

Transformation assays of TLS in the S. acidocaldarius chromosome. Diagrams A–D depict the transfer of the lesion to the Sulfolobus chromosome by OMT and its subsequent bypass. The transforming ssDNA (A) represents the antisense (minus) strand of the S. acidocaldarius pyrE gene. Near its midpoint, one nucleotide (solid symbol) corrects a point mutation in the recipient cell, while a DNA lesion replaces the adjacent nucleotide (“x”). This ssDNA is electroporated into recipient cells (B), where it anneals to a transient gap (Li et al. 2003). The resulting heteroduplex (B) is then replicated (C), which requires some form of bypass of the DNA lesion. Replication produces two completed chromosomes (i.e., sister chromatids) (D), but only daughter cells that retain the marker provided by the transforming ssDNA generate a colony. The remaining panels show the central section of each transforming ssDNA (bottom strand) opposite the recipient chromosome (top strand); numbers indicate positions within the pyrE-coding sequence. (E–I) The mutant codon of the recipient is underlined, with the corresponding amino acid shown to the right in bracketed italic type; the corresponding WT codon provided by the transforming DNA is given for the bottom strand. The selected nucleotide and query site (x) are shown in boldface type. (E–G) Sites in the pyrE gene of S. acidocaldarius. (H and I) The pyrE gene of S. solfataricus inserted into the S. acidocaldarius chromosome (see Table S1).

Figure 2

Dbh and bypass of 8-oxo-dG. The diagram depicts the most common responses to 8-oxo-dG (“oG” above) represented by bypass events in normal and Dbh-deficient mutants of S. acidocaldarius. Individual processes contributing to the bypass seen in dbh mutants were not resolved in this study and may include repair, error-free (recombinational) damage tolerance, or polymerase switching among B-family enzymes.