The constitutive association of Hat1p/Hat2p with ORC. (A) Co-immunoprecipitation (IP) of Hat1p with ORC from log-phase cells. Strains were: untagged control (JRY2334), Hat1–13myc (BSY676), and Orc5–13myc (BSY677). IPs were performed with monoclonal α-Orc3p and α-GFP antibodies. One twentieth of the extracts were loaded as whole cell extract (WCE). Immunoprecipitated Hat1–13myc was detected with mouse α-myc as a primary antibody (9E10). Note that equal amounts and concentrations of extracts were used for all IPs. (B) ORC binds Hat1p/Hat2p but not Hif1p. Strains were: untagged control, Hat1–13myc, Hat2–13myc (BSY691), and Hif1–13myc (BSY692). One twentieth of the extracts were loaded as WCE control. IPs were performed with monoclonal α-Orc3p and α-GFP control antibodies. (C) IP of Hat1p-13myc with α-Orc3p antibody in synchronized cells. Log-phase cells were either arrested in 3 μg/ml α-factor, 200 mM hydroxyurea, or 15 μg/ml nocodazole. Cells were also released from α-factor arrest and samples were taken at 20, 30, and 40 min after release (lanes 4–6). Lanes 7–12 show WCE controls. (D) FACS control of samples in panel C. Time after release is indicated (Rel).