Figure 2.

DDK phosphorylates Mcm2, Mcm4, and Mcm6 in the context of the pre-RC. (A) DDK phosphorylation of purified pre-RCs. Pre-RC assembly assays were performed with DNAs including wild-type (WT) or a mutant ARS1 lacking an ORC DNA-binding site (A−). DNA-associated proteins were treated with DDK or DDK-KD in the presence of [γ-³²P]ATP for 15 min at 25°C. (Left panel) Samples were separated by SDS-PAGE, and ORC, Cdc7, Dbf4, and Mcm2–7 proteins were detected by immunoblotting. (Right panel) The resulting blot was also analyzed by autoradiography, and the major phosphoproteins are labeled A–E. (B) Mcm2, Mcm4, and Mcm6 are phosphorylated by DDK. Pre-RCs were assembled with wild-type- and A-ARS1-containing DNAs using extracts in which the indicated Mcm subunit was epitope-tagged. An extract with untagged Mcm2–7 subunits was also tested. Pre-RCs were then treated with wild-type DDK kinase in the presence of [γ-³²P]ATP. Samples were analyzed by SDS-PAGE followed by immunoblotting and autoradiography. Arrows indicate the bands that were shifted when tagged with BCCP. Asterisks (*) indicate origin-independent phosphoproteins.