Fig 3.

ORCA association with ORC, Cdt1, and geminin is cell cycle regulated. (A) Association of ORCA with ORC, Cdt1, and geminin in human cells. Shown are HeLa nuclear extract fractionated over a Superdex 200 gel filtration column and fractions analyzed for ORCA, Orc2, Cdt1, and geminin by immunoblotting. Molecular mass markers are labeled above the blot. (Ba) ORCA immunoprecipitation during different stages of the cell cycle. ORCA associates with Orc2 throughout the cell cycle. ORCA associates with Orc1 during G₁ but not efficiently with the phosphorylated Orc1 during mitosis. ORCA associates with Cdt1 during G₁, as well as robustly with phosphorylated Cdt1 during mitosis. ORCA associates strongly with geminin from G₁/S to mitosis. (Bb) Immunoblot analysis of whole-cell extract from nocodazole-arrested mitotic extracts treated with phosphatase and untreated. Note that the phosphorylated form of Cdt1 and geminin collapses on phosphatase treatment. (C) Glycerol gradient sedimentation analysis of ORCA complex on material (asynchronous sample) immunoprecipitated using ORCA antibodies. The corresponding molecular mass markers are labeled. Note that ORCA and Orc2 cosediment in fractions 11 to 15, ORCA and Cdt1 cosediment in fractions 11 to 14, and ORCA and geminin cosediment in fractions 8 and 13 to 15. Fractions 13 and 14 contain ORCA, Orc2, Cdt1, and geminin, suggesting the existence of a quaternary complex, as well. (D and E) Glycerol gradient sedimentation analysis of ORCA-containing complex from G₁ extracts (D) and mitotic extracts (E). Note the cosedimentation of ORCA-Orc2-Cdt1 (fraction 11) in G₁ extracts and ORCA-Orc2-Cdt1-geminin (fractions 9 and 10) in mitotic extracts. (F) Gel filtration analysis of ORCA-containing complex during mitosis. (G) Sequential immunoprecipitation of ORCA, followed by Cdt1 from mitotic extracts. Immunoblot analysis of geminin corroborated the presence of ORCA-Orc2-Cdt1-geminin complex in mitotic extracts.