Azure c600 imager

OGG1 activity was determined using whole cell lysates as previously described [57 (link)], with minor modifications. Briefly, a 40-mer oligonucleotide containing an 8-oxoG at position 19 and labeled at the 3′ end with indodicarbocyanine (5′AGAGAAGAAGAAGAA(G*)AGATGGGTTATTCGAACTAGCCy5Sp/3′) was hybridized to its complementary sequence containing a cytosine opposite the 8-oxoG lesion (G*) by heating at 65 °C for 15 min, followed by cooling at room temperature for 3 h. Total protein was isolated from cells without addition of protease and phosphatase inhibitors. 100 µg of whole cell lysate was incubated with 200 nM 8oxoG:C duplex in assay buffer containing 200 mM Tris-HCl pH 7.5, 20 mM EDTA, 1 mg/mL BSA, and 5% glycerol. The reaction mixture was incubated for 3 h at 37 °C, and the excision reaction was stopped by adding equal volume (50 µL) of gel loading buffer II (AM8547, Thermo Fisher) and heating at 95 °C for 5 min. 10 µL of the reaction was loaded on a 20% polyacrylamide gel containing 8M urea in Tris-borate-EDTA buffer, pH 8.4 to separate the excised DNA from its substrate. Separated bands were visualized using Azure c600 imager (Azure biosystems, USA).

Two percent agarose gel electrophoresis with TAE buffer was used to separate PCR products using a 100 bp DNA ladder (TransGen Biotech, Beijing, China). An Azure C600 imager (Azure Biosystems, Dublin, CA, USA) was used to observe the bands. For Sanger sequencing, PCR amplification products were excised from the agarose gel and purified using a GeneJET Gel Purification Kit (Thermo Fisher Scientific, USA). The nucleotide sequences of the purified fragments were determined by Sanger sequencing using standard approaches by Geneseed (Guangzhou, China) [23 (link)].

To induce meiosis, diploid cells were grown in YP-acetate for 18 hours and then shifted to 1% potassium acetate sporulation medium at 1x10⁸ cells/ml. Following transfer to sporulation medium, 0.5 ml of culture was harvested at 0, 3, 5, 7, 9, 11, and 24-hour time points. Cells were disrupted in cold 16.6% trichloroacetic acid and 0.5mm glass beads (Biospec Products) in a Bullet Blue Blender for 5 minutes at speed setting 8 following the manufacturer’s instructions. Protein precipitates were washed in cold 95% ethanol, pelleted in a microcentrifuge, and resuspended in SDS-PAGE buffer. Samples were boiled for 10 minutes and pelleted at 16.4 x g for 15 minutes at 4°C. Equal volumes of each sample were loaded onto 8% polyacrylamide gels in duplicate. For one gel, blotted proteins were detected using primary antibodies against Zip1 (rabbit anti-Zip1 antibody raised against the C-terminal 264 amino acids of Zip1; used at a 1:3000 dilution) and goat anti-Rabbit horse radish peroxidase (HRP) conjugated antibody (Fisher PI31460; 1:5000 dilution). For the duplicate blot, Pgk1 was detected using mouse anti-Pgk1 (Molecular Probes 22C5; used at a 1:3000 dilution) and donkey anti-Mouse HRP secondary antibody (Jackson 715-035-151, 1:5000 dilution). Images were collected on an Azure c600 Imager.