Figure 7.
The dbf4-NΔ109 sld3-38A double mutant allowed late-origin firing in the presence of HU. (A) Wild-type and mutant cells were synchronized in G1 phase and released into medium containing 0.2 mM HU for the indicated times. Total protein extracts were examined by Western blotting for Rad53 to assess Rad53 activation (upper band). (B–D) RIs were separated by alkaline gel electrophoresis and detected by Southern blotting to measure the activity of early (ARS305) and late (ARS501 and ARS603) origins. Flow cytometry assays indicated all strains arrested in early S phase with HU (not shown); the budding indices are shown in E. (F) Wild-type Cdc7-Dbf4 or N-terminal Dbf4 truncation mutants were immunoprecipitated from asynchronous yeast extracts (t = 0) and after 1 or 2 hr exposure to 0.1 M HU, separated on an SDS gel, and then blotted for Cdc7 and Dbf4 proteins.