Figure 6.

Stu2 CEN binding is abolished in spc24-9 mutants, whereas Stu1 is still able to associate with CEN DNA. Multiplex PCR analysis of CEN1, PGK1, and CEN3 loci was performed with total chromatin (T) or immunoprecipitate (IP) as PCR templates. Strains were grown at 25°C to log phase and then either kept at 25°C or incubated at 30 or 37°C for 3 h. (A) Wild-type log phase cells grown at 30°C and carrying no epitope tag (No Tag; lanes 1 and 2), Stu1-Myc (lanes 3 and 4), and Stu2-Myc (lanes 5 and 6). (B) spc24-9 mutants carrying Stu1-Myc (lanes 1–4) and a wild-type strain with no tag at 37°C (lanes 5 and 6). (C) Stu2-Myc in a wild-type (lanes 1 and 2), spc24-9 (lanes 3 and 4), and an untagged wild-type strain (No Tag; lanes 5 and 6). Strains were grown to log phase at 25°C, HU was added to a final concentration of 0.2 M HU, and cells were shifted to 30°C for 3 h. (D) Stu2-Myc in a wild-type (lanes 1–6) and spc24-9 (lanes 7–12) strains. No Tag strain (lanes 13–18) is an untagged wild-type strain. For all ChIP assays where the spc24-9 mutant was used, we included both a wild-type and spc24-9 untagged control, and we saw similar results; thus, only the wild-type untagged control is presented.