Mutational analysis of Scm3. A, schematic diagrams of wild-type Scm3 and site-directed Scm3 mutants. The NES, conserved motif (CD), potential NLS and C-terminal acidic (D/E) regions are boxed. B, plasmid shuffle complementation tests of mutants shown in A. Growth on 5-fluroorotic acid (FOA) medium indicates the respective mutant allele provides Scm3 function.

Evolutionarily conserved core motif of Scm3 is required to load Cse4 at the centromere but not Ndc10. Strains were constructed in which the endogenous copy of Scm3 was under control of the Gal promoter, a plasmid contained another source of Scm3, and Cse4 was tagged with 12myc epitopes. In galactose-containing medium, Scm3 is expressed (A, C, and E) but in glucose (B, D, and F) the only source of Scm3 is the plasmid. The ChIP/qPCR from the galactose cultures serves as a control. ChIP/qPCR shows that Cse4 is not present at CEN1 in the Scm3-I110H mutant background in glucose in either asynchronous cultures (B) or at CEN3 in G₁-arrested and released (4 h) cultures (D). Error bars represent ± the average deviation of biological replicates. A control ChIP omitting antibody was performed for each sample; all values were below 0.01 (ratio of no antibody/total chromatin). E and F, ChIP/qPCR for Ndc10 in G₁-arrested and released (4 h) cultures shows that Ndc10 is present at CEN3 in both the lethal mutant backgrounds. See also supplemental Fig. 1 for cytometry profiles.

Scm3 is a Cse4-specific nucleosome assembly factor. A, Scm3-mediated chromatin assembly reaction was performed by incubating the relaxed plasmids and yeast canonical octamers at a ratio of 1:1 with increasing amounts of Scm3 (ratio of 1.0, 1.2). Higher amounts also had no effect. B, chromatin assembly was performed on relaxed plasmids with Nap1 and yeast canonical octamers as in A (ratio of 1.0, 1.2). C, diagram of the H3/Cse4 chimeric protein is shown. Chromatin assembly reactions were performed on the relaxed pCEN1-10X plasmid with increasing amounts of reconstituted octamers containing H3/Cse4 chimeric protein and Scm3 or Nap1. D and E, H2A/H2B are critical for Scm3 to induce supercoiling. Chromatin assembly reactions were performed under the conditions in Fig. 4B by incubating the relaxed pG5E4-5S and pCEN1-10X plasmid with (D) increasing amounts of reconstituted Scm3/Cse4/H4 complex alone (His₆-Scm3/Cse4/H4) or (E) Scm3/Cse4/H4 complex with an equivalent molar ratio of H2A/H2B dimers. In the lane labeled dimer, H2A/H2B dimers were added at a ratio of 0.8 to the DNA. See also supplemental Figs. 2 and 3.

Co-immunoprecipitation of Scm3 mutants with Cse4 and Ndc10. Immunoprecipitations of Scm3-FLAG mutants were performed in a background containing Cse4-Myc or Ndc10-Myc. No Tag indicates that Scm3 does not have the FLAG tag. Western blotting was carried out with anti-Myc antibody. Full-length Scm3-FLAG often runs as a doublet for reasons that are currently unclear. A, evolutionarily conserved motif is required for Cse4 interaction. Point mutants in this motif no longer interact with Cse4. When the 25 N-terminal amino acids of Scm3 are deleted, this protein still pulls down Cse4. B, both point mutants in the conserved motif and a mutant with a deletion of 25 amino acids from N terminus still co-immunoprecipitate with Ndc10.

Scm3 can assemble Cse4-containing nucleosomes in vitro. Nucleosome assembly activity of Scm3 was studied with a plasmid supercoiling assay. Supercoiled plasmids were purified from E. coli (S) and relaxed by addition of topoisomerase I (R). Octamers alone (Oct only) was included as a control for each assembly experiment. A, schematic diagram of plasmid construct pG5E4-5S and pCEN1-10X plasmids is shown. B, chromatin assembly was performed by incubating the relaxed plasmids with purified His₆-Scm3 and Cse4 octamers. DNA and Cse4 octamer amounts are held constant at a ratio of 1:1, and Scm3 is added at a ratio of 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6. C, Nap1 was incubated with Cse4 octamers. DNA and Cse4 octamer amounts are held constant at a ratio of 1:1, and Nap1 is added at a ratio of 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0. D, control assembly reaction with only His₆-Scm3 at a ratio to DNA of 0.6 and 0.8 does not yield any supercoils on either of the plasmids. Higher amounts also had no effect. E and F, conserved core of Scm3 is necessary for chromatin assembly. Chromatin assembly reactions were performed by incubating the relaxed pG5E4-5S or pCEN1-10X plasmid with Cse4 octamers and either Scm3-Δ25N (E) or Scm3-I110H (F).