FIG. 8.

The interaction between Dpb11 and Ddc1 requires a functional Mec1 kinase. Plasmids pFP1 (pJG4-5-DPB11) and pFP2 (pEG202-DDC1) were cotransformed with pSH18-34, a β-galactosidase reporter plasmid, in either MEC1 or mec1-1 mutant yeast cells. A similar strategy was adopted for pFP4 (pEG202-ddc1-C), which carries only the C-terminal fragment (nucleotides 309 to 612) of DDC1, containing the 11 putative Mec1 phosphorylation target sites and for pFP10 (pEG202-ddc1M8). To assess two-hybrid interaction, these strains were patched onto 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) plates containing either raffinose (Raf; Dpb11 prey repressed) or galactose (Gal; Dpb11 prey expressed) as a carbon source. After 3 days, the plates were analyzed. The strains in panel A are YFP50 (MEC1, top), YFP52 (MEC1, bottom), YFP113 (mec1-1, top), and YFP114 (mec1-1, bottom). A positive control in the mec1-1 mutant strain (p53 versus large T antigen [TAg]) was also used. The strains in panel B are, from left to right, YFP50, YFP86, YFP54, and YFP153.