(A) A diagram of a 190-bp CEN region. It consists of CDEI (small box), CDEIII (large box), and CDEII between the boxes. The conserved CDEIII sequence with two single base pair substitutions that abolish centromere functions are shown. (B) The CEN3 minichromosome construct used in Figure 3 (a gift from S. Biggins and T. Tsukiyama). This ~2-kb minichromosome contains a TRP selectable marker (Dotted arrow indicates the location of ORF), CEN3 (solid arrow) and a short stretch of bacterial DNA (green bar), which was used as the Southern blot probe target, because it does not cross-hybridize with yeast genomic DNA (data not shown). The diagram is approximately to scale. The known locations of H3 nucleosomes that were previously mapped (Thoma et al., 1984) are shown as blue circles. Red circles indicate the presumed locations of Cse4p nucleosomes. We do not know whether a nucleosome is present over the bacterial sequence used for Southern blotting (dashed circle). Expected numbers of H3 and Cse4p nucleosomes (assuming that there are no nucleosomes corresponding to the dashed circle) are indicated. The relatively low nucleosome density of 2000 bp/8 nucleosomes = 250 bp/nucleosome (yeast average: 165 bp/nucleosome (Nelson and Fangman, 1979)) results from the presence of several well-positioned nucleosome and several nucleosome-free regions on the minichromosome. (C) The CEN3+CEN6 double centromere minichromosome construct used in Figure 4 (See Table S1). (D) same as (C) except that CEN6 carries the 2-bp substitution shown in (A). (E) same as (C) except that both CEN3 and CEN6 carry the 2-bp substitution shown in (A).

(A) Minichromosomes containing 2, 1 or no functional centromeres (# of CENs) that were derived from wildtype (WT), ndc10-1, or ndc80-1 strains were allowed to go through S phase at either 25°C or 37°C. Total DNA were isolated and electrophoresed on an agarose gel containing 0.3µg/ml chloroquine to resolve topoisomers. Southern blot analysis was performed to detect the minichromosomes. The most slowly migrating band contains both a nicked circle (N) and a topoisomer with no net writhe (0). The numbers correspond to the value of net writhe in this chloroquine gel. When a relaxed circle was run in an identical chloroquine gel, its peak distribution was +2 and +3, which co-migrates with −2 and −3 topoisomers (data not shown). Note that the first detectable band in lanes 5, 6, 11, 12, 17, and 18 corresponds to −3. The Southern blot shown here is a representative of independent experiments. (B) Densitometric trace of (A) with lane numbers as indicated. The asterisks mark the nicked and “0” topoisomer in (A). The double asterisks in the trace of lane 18 indicate a smudge in Figure 4A, which was not included in the analysis provided in Table 2.

(A) An ~7 kb plasmid containing an ~3 kb Drosophila satellite DNA insert (Furuyama et al., 2006) electrophoreses as negatively supercoiled species in an agarose gel after isolation from E.coli (S). The plasmid DNA was relaxed by topoisomerase (R), and H3 chromatin was assembled in vitro (34AB) in the presence of the Drosophila RbAp48 chaperone, as previously described. The top panel shows the topoisomer separation on an agarose gel without chloroquine, whereas the bottom panel shows their migration in the presence of 1µg/ml chloroquine. Gels were stained with ethidium bromide after separation to visualize DNA. The slower migration of topoisomers induced by H3-containing nucleosome relative to the migration of relaxed DNA (R) indicates that H3-nucleosomes induce negative supercoils (red bracket). (B) same as in (A), except that CID (Drosophila CenH3) was used in place of H3 (C4AB). The faster migration of topoisomers induced by CID-containing nucleosome relative to the migration of relaxed DNA (R) in the presence of chloroquine indicates that CID-nucleosomes induce positive supercoils in vitro. (C) Identical chromatin assembly reactions were performed as in (B) using four independent plasmids containing randomly cloned 3-kb Drosophila genomic DNA (Clone #1 through #4; See also Figure S1). CID nucleosomes induce positive supercoils regardless of DNA sequence, as evident from migration patterns that are similar to those in (B). S: negatively supercoiled; R, relaxed; C: CID chromatin.

Total DNA was isolated from either a wildtype (WT) or a mutant ndc10-1 strain carrying the same CEN3 minichromosome (Figure 2B), released from α-factor arrest at the restrictive temperature (37°C), and purified DNA was resolved on an agarose gel containing 0.3µg/ml chloroquine. A Southern blot to detect the minichromosome is shown on the left. A densitometry trace of each lane is shown on the right. The most slowly migrating band contains both a nicked circle (N) and a topoisomer with no net writhe in the presence of chloroquine (0). The numbers correspond to the value of net writhe in this chloroquine gel. The minichromosome has fewer negative supercoils in the wildtype strain with Cse4p present at the centromere, compared to the ndc10-1 mutant strain, which loses Cse4p at the restrictive temperature. The arrow in the trace indicates the location of the mean topoisomer distribution. The asterisks mark the bands containing nicked and “0” topoisomer, which was omitted from calculating the mean distribution. Nicked and ”0” topoisomer were resolved by two-dimensional electrophoresis, where similar results were obtained (Figure S4).

A) Crystal structure of a nucleosome (Luger et al., 1997). One H3/H4 dimer is colored in light blue, the second H3/H4 dimer is colored in light red, both H2A/H2B dimers are shown in gray, and DNA is shown in brown wrapping around the histone octamer. A close-up view of the N-terminal helix of H3 (red stick) and H2A docking domain (magenta) with their side chains shown in the top inset. The interaction between two H2A molecules at the bottom of the octameric structure through their Loop 1 (green) is evident in the bottom inset. B) The structure in A) is rotated 90° to emphasize the spiral of 4 histone dimers wrapping DNA in the left-hand orientation. C) and D) Cartoons of left- and putative right-handed octamers. The two H3/H4 dimers were differentially colored in light blue and magenta, while the two H2A/H2B dimers are shown in gray. The DNA double helix is shown as a black line. Black ovals in C) depict the interaction between the N-terminal helix of H3 and the docking domain of H2A. Blue dots indicate H2A Loop1. In the right-handed octamer, which does not exist, the indicated interaction surfaces would face away from each other. E) One half of the left-handed structure shown in B), illustrating exposed surfaces in the absence of the other half. F) a cartoon of what a right-handed structure might look like. F) was produced using Abobe Photoshop, maintaining the approximate orientation of H4/H2B 4-helix bundle.