FIG. 5.
Cell cycle-regulated interaction of Pol1p with Spt16p and Ctf4p. Cells harboring POL1-TAP, SPT16-13Xmyc, and CTF4-3XHA were synchronized in G1 with α-factor and then released into YPD medium at 22°C. Cell samples were removed at the indicated times for FACS analysis and immunoprecipitation. (A) Flow cytometry profiles of cells at indicated times. (B) Immunoprecipitations of POL1-TAP were performed from cell extracts of the wild type and the pol1-1 mutant with rabbit IgG-agarose at the indicated times (in minutes). Coprecipitation of Spt16p-13Xmyc and Ctf4p-3XHA with Pol1p-TAP were detected with anti-myc and anti-HA antibodies, respectively. Control lanes, Western blot immunoprecipitates with rabbit IgG-agarose from extracts of cells that do not harbor the TAP-tagged POL1 but contain SPT16-13Xmyc or CTF4-3XHA with anti-myc (9E10) or anti-HA (12CA5). (C) Immunoprecipitations of TAP-tagged Pol1p were performed from cell extracts of the wild type and the pol1-1 mutant by rabbit IgG-agarose at the indicated times, as in panel B. Coprecipitation of Spt16p-CFP with Pol1p-TAP was detected with anti-GFP. Control lanes show Western blot immunoprecipitates with rabbit IgG-agarose from extracts of cells that do not harbor the TAP-tagged POL1 or pol1-1 genes but that contain SPT16-CFP with anti-GFP. Protein loading control was performed by Western blotting immunoprecipitates with anti-IgG.