FIGURE 5.

FHA-mediated dimerization is important for the DNA damage functions of Mdb1. A, FHA-mediated dimerization is important for the genotoxin sensitivity induced by Mdb1 overexpression. Cells were pregrown in a thiamine-free medium for 20 h to allow full induction of the Pnmt1 promoter, and 5-fold serial dilutions were spotted on EMM-based thiamine-free plates. The strains used were DY15615, DY15616, DY17216, DY17218, and DY17220. B, the expression levels of different versions of Mdb1 analyzed in A. Coomassie staining of PVDF membrane after immunodetection was used to assess protein loading level and blotting efficiency (43). C, adding GST tag allowed Mdb1(105–624) to self-interact. The strains used were DY24566 and DY24567. D, adding LZ tag allowed Mdb1(105–624) to self-interact. The strains used were DY24570 and DY24571. E, FHA-mediated dimerization is required for the ability of Mdb1 to rescue the CPT sensitivity of mdb1Δ crb2-F400A double mutant. The expression was under the control of the P81nmt1 promoter. Cells were pregrown in a thiamine-free medium for 20 h before being spotted on CPT-containing thiamine-free plates. The strains used were DY16581, DY17124, DY17126, DY16579, and DY16573. IB, immunoblot.