Figure 1
The four HRR genes, herA , mre11 , rad50 , and nurA , which are encoded in a single operon, are all essential for cell viability. (A) Schematic diagram depicting the construction of the strain used in the gene knockout analysis. Marker insertion and target gene deletion (MID) and a subsequent mutant propagation assay were utilized. The star indicates a putative spontaneous mutation site in pyrEF. (B) Mutant propagation assay for pyrEF-lacS-integrated transformants (pMID-herA-T, pMID-mre11-T, pMID-rad50-T, and pMID-nurA-T). orc1-3, a nonessential gene coding for one of the three replication initiation proteins, was used as a positive control, whereas REY15A is a reference for pyrEF mutation. Strains were cultivated in STVy medium supplemented with 2 mg/ml uracil. The counter selection chemical, 5-FOA, was added when OD600 reached 0.4. The culture was diluted with fresh medium to OD600 ~ 0.1 when the cells had grown to early stationary phase. Absorbance of the culture was measured.