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

Abstract

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.

Keywords: DNA polymerase; DNA repair; DNA replication; Sulfolobus acidocaldarius; hyperthermophilic archaea.

FIGURE 1

Growth after UV-B irradiation (A). Each overnight culture of the deletion strains was irradiated with UV for 80 s (1600 J/m²) and cultivated at 75°C. +UV represents a UV-treated sample. The error bars indicate the mean ± SD calculated using two biological replicates. Closed circle, the growth of DP-1; closed square, the growth of ΔpolB2 (HM-1); closed diamond, the growth of ΔpolB3 (HM-2); closed triangle, the growth of Δdbh (HM-3); open circle, the growth of ΔpolB2ΔpolB3 (HM-4); opened square, the growth of ΔpolB2Δdbh (HM-5); open diamond, the growth of ΔpolB3Δdbh (HM-6); open triangle, the growth of ΔpolB2ΔpolB3Δdbh (HM-7). The deletion strains were tested for UV sensitivity (B,C). After UV-B exposure (15 s), diluted samples (10⁰–10^{– 6}) of DP-1 and the deletion strains were spotted onto XTU plates and cultivated at 75°C. (B) mock-treated samples; (C) UV exposure for 15 s.

FIGURE 2

Growth in the presence of DNA-damaging agents. Overnight cultures of each deletion strain and DP-1 were inoculated into liquid medium in the presence of DNA-damaging agents {cisplatin [40 (A) and 20 μg/mL (B)] and 4-NQNO [0.8 (C) and 0.4 μg/mL (D)]} and cultivated at 75 and 60°C, respectively. +A represents the growth curve in the presence of DNA-damaging agents. The error bars indicate the mean ± SD, calculated using two biological replicates. Closed circle, the growth of DP-1; closed square, the growth of ΔpolB2 (HM-1); closed diamond, the growth of ΔpolB3 (HM-2); closed triangle, the growth of Δdbh (HM-3); open circle, the growth of ΔpolB2ΔpolB3 (HM-4); open square, the growth of ΔpolB2Δdbh (HM-5); open diamond, the growth of ΔpolB3Δdbh (HM-6); open triangle, the growth of ΔpolB2ΔpolB3Δdbh (HM-7).

FIGURE 3

MMC sensitivity. After MMC treatment (10 mM), diluted samples (10⁰–10^{– 6}) of DP-1 and the deletion strains were spotted onto XTU plates and cultivated at 75°C. (A) mock-treated samples; (B) MMC-treated samples.

FIGURE 4

MNNG sensitivity. After MNNG treatment (100 μg/mL), diluted samples (10⁰–10^{– 6}) of DP-1 and the deletion strains were spotted onto XTU plates and cultivated at 75°C. (A) mock-treated samples; (B) MNNG-treated samples.

FIGURE 5

Heat-shock sensitivity. After treatment at 90°C (0, 2, 3, and 4 min), diluted samples (10^{– 6}–10⁰) of DP-1 and the deletion strains were spotted onto XTU plates and cultivated at 75°C. (A) mock-treated samples; (B–D) heat-shock for 2, 3, and 4 min at 90°C, respectively.

FIGURE 6

Cultivation time to reach OD₆₀₀ = 0.1. Overnight cultures of the deletion strains and DP-1 were inoculated into liquid medium in the presence of a DNA replication inhibitor {novobiocin (1.2 μg/mL) (A) and HU [0.075 (B) and 0.1 mM (C)]} and cultivated at 75 and 60°C. The cultivation time was calculated from the growth curves of the deletion strains in the presence of a DNA replication inhibitor. White and gray bars indicate cultivation in the absence and presence of the DNA replication inhibitor, respectively. The error bars indicate the mean ± SD, calculated using two biological replicates.