FIG. 6.

Assembly of Pol1p onto late-replicating origin is delayed in the pol1-1 mutant. (A) Flow cytometry profile of the asynchronous wild type and the pol1-1 mutant harboring POL1-TAP or pol1-1-TAP at different temperatures. (B) Assembly of wild-type and mutant Pol1p-TAP onto replication origins (ARS). Wild-type and pol1-1 mutant cells containing the TAP-tagged POL1 or pol1-1 were synchronized by α-factor arrest and then released into YPD medium at 22°C. The cells were withdrawn from the culture every 10 min for FACS analysis and CHIP assay with rabbit IgG-agarose. A PCR was performed on the immunoprecipitates and on whole-cell extract as input control at each time point. (C) Checkpoint kinase Rad53p is not activated in the pol1-1 mutant. Wild-type cells harboring POL1-TAP RAD53-13Xmyc or mutant cells harboring pol1-1-TAP RAD53-13Xmyc were grown at 22 and 25°C with or without 0.2 M hydroxyurea. The whole-cell extract was then subjected to electrophoresis fractionation and Western blotting with anti-myc antibody to detect the mobility of Rad53p phosphorylation.