Roles of the Y-family DNA polymerase Dbh in accurate replication of the Sulfolobus genome at high temperature

Abstract

The intrinsically thermostable Y-family DNA polymerases of Sulfolobus spp. have revealed detailed three-dimensional structure and catalytic mechanisms of trans-lesion DNA polymerases, yet their functions in maintaining their native genomes remain largely unexplored. To identify functions of the Y-family DNA polymerase Dbh in replicating the Sulfolobus genome under extreme conditions, we disrupted the dbh gene in Sulfolobus acidocaldarius and characterized the resulting mutant strains phenotypically. Disruption of dbh did not cause any obvious growth defect, sensitivity to any of several DNA-damaging agents, or change in overall rate of spontaneous mutation at a well-characterized target gene. Loss of dbh did, however, cause significant changes in the spectrum of spontaneous forward mutation in each of two orthologous target genes of different sequence. Relative to wild-type strains, dbh(-) constructs exhibited fewer frame-shift and other small insertion-deletion mutations, but exhibited more base-pair substitutions that converted G:C base pairs to T:A base pairs. These changes, which were confirmed to be statistically significant, indicate two distinct activities of the Dbh polymerase in Sulfolobus cells growing under nearly optimal culture conditions (78-80°C and pH 3). The first activity promotes slipped-strand events within simple repetitive motifs, such as mononucleotide runs or triplet repeats, and the second promotes insertion of C opposite a potentially miscoding form of G, thereby avoiding G:C to T:A transversions.

Figure 1

Construction of dbh⁻ strain CS2 Panel A. Cloning and disruption of dbh. Diagram shows major features of construct pS42, containing the disruption of dbh (Saci_0554) that was transferred to the S. acidocaldarius genome. For details of construction, see Materials and Methods. Panel B. Confirmation of dbh disruption. PCR products amplified from genomic DNA of wild-type S. acidocaldarius (lanes1 and 3) and dbh disruption mutants (lanes 2 and 4). Lanes 1 and 2 show amplification of the whole gene and lanes 3 and 4 show amplification of an internal portion of the gene. The primers used to amplify the whole gene were Saci0554f1 and Saci0554r1 and the primers used to amplify the internal portion were Saci0554f3 and Saci0554r3 (see Table 1). MW: molecular weight marker (bacteriophage λ DNA digested with BstEII); fragment lengths in bp are shown in the left-hand margin.

Figure 2

Sensitivity to killing by UV or cisplatin Panel A shows the fraction of cells surviving the indicated fluence of UV-C; error bars indicate standard deviations. Panel B shows the fraction of cells surviving overnight room-temperature treatment of cisplatin at the indicated concentration (μg/ml); error bars indicate standard errors. Symbols: circles, strain CS2; triangles, strain DG250; squares, strain DG185; diamonds, strain DG251.

Figure 3

Spontaneous pyrE mutation spectra in disruptant and control strains. The sequence of the S. acidocaldarius (A and B) or S. solfataricus (C and D) pyrE coding region with markings indicating the spontaneous mutations recovered from (A) CS2 (B) DG185 (C) DG 250 (D) DG 251. BPSs are denoted with an A, T, C, or G above a base in the sequence. Deletion mutations (> 3bp) are represented by bold italicized text. Tandem duplications (> 6bp) are indicated by an underline or overline. A ‘+’ or a ‘Δ’ sign above the sequence denotes a small insertion or deletion (< 6bp), respectively, of the base(s) below it. Placement of these symbols above mono-nucleotide runs is arbitrary, as all positions of a run are genetically equivalent. Single base pair insertions that differ from their adjacent bases is shown by a ‘v’ sign positioned in between two bases on the sequence with the insertion above it. Bracketed text with a ‘+’ or ‘Δ’ indicates insertions or deletion mutations, respectively, occurring in a single mutant.

Figure 3

Spontaneous pyrE mutation spectra in disruptant and control strains. The sequence of the S. acidocaldarius (A and B) or S. solfataricus (C and D) pyrE coding region with markings indicating the spontaneous mutations recovered from (A) CS2 (B) DG185 (C) DG 250 (D) DG 251. BPSs are denoted with an A, T, C, or G above a base in the sequence. Deletion mutations (> 3bp) are represented by bold italicized text. Tandem duplications (> 6bp) are indicated by an underline or overline. A ‘+’ or a ‘Δ’ sign above the sequence denotes a small insertion or deletion (< 6bp), respectively, of the base(s) below it. Placement of these symbols above mono-nucleotide runs is arbitrary, as all positions of a run are genetically equivalent. Single base pair insertions that differ from their adjacent bases is shown by a ‘v’ sign positioned in between two bases on the sequence with the insertion above it. Bracketed text with a ‘+’ or ‘Δ’ indicates insertions or deletion mutations, respectively, occurring in a single mutant.