FIG. 6.

Phosphorylation of the Mcm4 protein on the replicative chromatin. (A) Demembranated sperm nuclei were incubated in a cytosolic high-speed extract (HSS) or interphase extracts (LSS) containing 50 μg of aphidicolin per ml. When indicated, the cytosolic extract was supplemented with a 1/10 volume of a membrane fraction, and the interphase extract was supplemented with either 3 mM DMAP or 0.5 mg of wheat germ agglutinin (WGA) per ml. After a 90-min incubation at room temperature, the chromatin was isolated and the Mcm4 isoform associated with the chromatin was identified by Western blot analysis. (B) Demembranated sperm nuclei were incubated in interphase extracts containing 50 μg of aphidicolin per ml and 1 μCi of [γ-³²P]ATP per μl of extract. After a 1-h incubation at room temperature, the chromatin was separated from the cytosol. Mcm proteins were immunoprecipitated from the cytosolic and from the chromatin fractions using anti-Mcm4 antibodies. Western blot analysis and autoradiography identified the phosphorylated Mcm subunits. Total chromatin was also analyzed. (C) Interphase extract was incubated with or without 0.5 μM p21-Cip recombinant protein (40) for 10 min at room temperature. Sperm nuclei, aphidicholin, and [γ-³²P]ATP were then added to the extract and incubated for an additional hour. Phosphorylated Mcm proteins on the chromatin were purified as in panel B.