FIGURE 6.
Analysis of DSB repair at the PHO5 gene under transcriptionally inactive and active conditions. A, schematic diagram for the experimental strategy. Inactive and active refer to DSB repairs under transcriptionally inactive and active conditions, respectively. B and C, the yeast strain RSY33 was initially grown in YPG-Pi up to an A600 of 0.4 and then switched to YPD (denoted as +Pi) or YPD-Pi (denoted as -Pi) for 1, 2, 3, and 4 h. Genomic DNAs from these yeast cultures were prepared and analyzed by PCR for DSBs at the PHO5 locus using the primer pair flanking the Ho cut site. A specific region within SMC2 was amplified as control using the same genomic DNAs. The PCR signals at the 0 h time point for the PHO5 and SMC2 loci were set to 100, and the PCR signals at 1, 2, 3, and 4 h were normalized with respect to 100. The ratios of normalized PCR signals at 1, 2, 3 and 4 h at the PHO5 Ho cut site to those at SMC2 are plotted in the form of a histogram. A ratio that is greater than 1 would indicate the DSB repair.