Figure 1.

DDK phosphorylates the C terminus of Rad18. (A) Recombinant Rad18–Rad6 complex was incubated with purified Cdc7-Dbf4 in the presence of [³²P]γATP. Reaction products were resolved by SDS-PAGE and analyzed by autoradiography (top) and Coomassie staining (bottom). The Rad18 and Rad6 bands were excised from the dried gel, and the radioactivity of each band was measured by scintillation counter. The incorporation of phosphate per molecule of Rad18 protein was calculated on the basis of the specific radioactivity of [³²P]γATP in the kinase reactions. (B) Recombinant Rad18–Rad6 complex was incubated with purified DDK complexes containing WT Cdc7 or a kinase-dead (KD) Cdc7 K90 → E mutant in the presence of [³²P]γATP. Reaction products were analyzed as described for A. (C) The indicated regions of hRad18 were expressed as recombinant GST fusion proteins in E. coli. Expression of the GST fusion proteins was validated by immunoblotting with anti-GST. GST fusions of Rad18 were tested for in vitro phosphorylation by Cdc7-Dbf4. Molecular mass is indicated in kilodaltons next to the gel blots.