FIG. 3.

Dbf4 motif M is required for normal cell growth, while motif N confers resistance to both HU and MMS. (A) DY-2 cells were transformed with pCM190-Dbf4-FL, -Dbf4ΔM, -Dbf4ΔN, -Dbf4Δ110-296, -Dbf4(1-296), or empty vector pCM190 (V). A complementation assay was performed as described in Materials and Methods. Cells were grown either in the presence (+Dox) or absence (-Dox, entire row) of Dox, which represses pCM190-based expression, at the indicated temperatures. Western blot analysis to confirm plasmid-based expression was conducted using a mouse monoclonal anti-Myc antibody (Sigma) and Alexa Fluor 488 goat anti-mouse immunoglobulin G polyclonal secondary antibody (Invitrogen). (B) DY-2 cells transformed with pCM190-Dbf4-FL (FL) or pCM190-Dbf4ΔM (ΔM) as described above were further transformed with either pJG 4-6 (V) or pJG-Cdc7 (Cdc7) and grown in the presence or absence of Dox, as indicated, at 23°C. (C) Tetrad dissection of DY-75 (ΔM/+) and DY-76 (ΔN/+) strains. Gene replacement, sporulation, and tetrad analysis were performed as described in Materials and Methods. Growth of spores following dissection of four tetrads corresponding to each strain is shown. (D) A 10-fold dilution series of the Dbf4ΔN and isogenic wild-type haploid strains at a starting concentration of 1 × 10⁷ cells/ml was spotted onto YPD plates containing the indicated concentrations of MMS and HU. Plates were incubated at 30°C for 2 days.