Pbr322

Topo I and pBR322 were obtained commercially from Takara Bio Inc. (Shiga, Japan). And the enzyme inhibitory activity was determined by our previous methods [19 (link)].

The Topo I enzyme inhibitory activity was determined by measuring the conversion of supercoiled pBR322 DNA to relaxed isomers. Topo I and pBR322 were purchased from Takara Bio Inc., Tokyo, Japan. The reaction system (20 μL) was mixed with 2 μL reaction buffer solution, 0.5 μg pBR322 DNA, 2 μL of different compounds, 1 μL One unit of Topo I, and distilled water added to 20 μL, and this was reacted at 37 °C for 30 min. The reaction was terminated by adding 0.5% SDS, 0.25 μg/mL bromophenol blue and 15% glycerol. The reaction products were subjected to 0.8% agarose gel at 90 V for 50 min at room temperature, which was stained with 5 μg/mL ethidium bromide, after which imaging was conducted by the Bio-Rad Gel Doc^TM XR+ system, Image Lab, California, CA, USA.

To generate plasmids expressing short hairpin RNA (shRNA) against TPM2 (sh1-TPM2 and sh2-TPM2), a pair of oligos (5’-ACC GGC AGA GAA ATC TGC ATT CTA TTG TTA ATA TTC ATA GCA ATA GAA TGC AGA TTT CTC TGT TTT-3’ and 5’-CGA AAA AAC AGA GAA ATC TGC ATT CTA TTG CTA TGA ATA TTA ACA ATA GAA TGC AGA TTT CTC TGC-3’ for sh1-TPM2; 5’- ACC GGG TAT TCT GAA TCT GTG AAG GAG TTA ATA TTC ATA GCT CCT TCA CGG ATT CAG AAT ACT TTT-3’ and 5’CGA AAA AAG TAT TCT GAA TCC GTG AAG GAG CTA TGA ATA TTA ACT CCT TCA CAG ATT CAG AAT ACC-3’ for sh2-TPM2) were annealed and inserted into the BbsI sites of pRSI9-U6-(sh)-UbiC-RFP-2A-Puro (Cellecta). To generate an HBV-expressing plasmid (pHBI), a 1.5-fold HBV genome (accession number X01587) (Fujiyama et al., 1983 (link)) was cloned into pBR322 (Takara). The full-length TPM2 transcript (accession number XM_017015092) was cloned into pLV-SIN-CMV-Hyg (Takara).

For UV damage tests of plasmid DNA pBR322 (Takara Bio, Kusatsu, Japan) by irradiation at 222 and 254 nm, E. coli HB101 competent cells Quick DH5α (DNA-913) were purchased from TOYOBO (Osaka, Japan). Upon UV exposure on DNA 0.1 μg/mL in TE buffer solutions (pH7.5, 1 mM Tris–HCl/0.1 mM EDTA), E. coli HB101 cells were transformed with the plasmid DNA and cultured on LB agar plates containing ampicillin (50 μg/mL, Nacalai Tesque, Kyoto, Japan).

To determine the genome type of halovirus VOLN27B, the genome of VOLN27B was isolated from the virus stock according to the method described by Summer [23 (link)] using Wizard DNA Clean-Up System (Promega, USA). Genomic DNA (1 μg) was digested with DNase I (5 U) (TaKaRa, Japan), Exonuclease III (20 U) (TaKaRa, Japan), and Mung Bean Nuclease (4 U) (TaKaRa, Japan) at 37°C for 1 h, and the resulting product was checked by DNA electrophoresis. An equal amount of undigested plasmid pBR322 (NEB, USA) was used as a circular dsDNA standard, and the EcoRI (TaKaRa, Japan) cleaved pBR322 was used as a linear dsDNA standard. These untreated and EcoRI leaved pBR322 plasmids were also used as controls in treatments nucleases DNase I, Exonuclease III, and Mung Bean Nuclease.

Supercoiled pBR32 DNA was used as a substrate to determine the Topo I catalytic activity. Commercial samples of Topo I and pBR322 were obtained from Takara Bio Inc. The buffer solutions were prepared using 500 mM KAc, 200 mM Tris-Ac, 100 mM Mg(Ac)₂, and 1 mg/mL BSA. The procedure used for determination of enzyme inhibitory activity was similar to one reported by Xu et al. [27 (link)].

PpIX was purchased from MP Biomedicals (Illkirch, France). Aminophenyl fluorescein (APF) was purchased from Goryo Chemical (Sapporo, Japan). Dihydroethidium (DHE), dimethyl sulfoxide (DMSO), methanol, ethanol, PI, and PBS buffer were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). SOD was purchased from MP Biomedicals (Eschwege, Germany). φX174 Hae III digest and pBR322 (Accession number: GenBank J01749.1) were purchased from Takara Bio (Kusatsu, Japan). A DNA damage detection kit (containing γH2AX monoclonal antibody and secondary antibody) was purchased from Dojindo Laboratories (Kumamoto, Japan).