Histone H3-variant Cse4-induced positive DNA supercoiling in the yeast plasmid has implications for a plasmid origin of a chromosome centromere

Abstract

The Saccharomyces cerevisiae 2-μm plasmid is a multicopy selfish genome that resides in the nucleus. The genetic organization of the plasmid is optimized for stable, high-copy propagation in host-cell populations. The plasmid's partitioning system poaches host factors, including the centromere-specific histone H3-variant Cse4 and the cohesin complex, enabling replicated plasmid copies to segregate equally in a chromosome-coupled fashion. We have characterized the in vivo chromatin topology of the plasmid partitioning locus STB in its Cse4-associated and Cse4-nonassociated states. We find that the occupancy of Cse4 at STB induces positive DNA supercoiling, with a linking difference (ΔLk) contribution estimated between +1 and +2 units. One plausible explanation for this contrary topology is the presence of a specialized Cse4-containing nucleosome with a right-handed DNA writhe at a functional STB, contrasted by a standard histone H3-containing nucleosome with a left-handed DNA writhe at a nonfunctional STB. The similarities between STB and centromere in their nucleosome signature and DNA topology would be consistent with the potential origin of the unusual point centromere of budding yeast chromosomes from the partitioning locus of an ancestral plasmid.

Fig. 1.

Reporter plasmids and protocols for cell-cycle arrest and release. (Upper) The reporter plasmids pSTB-CEN4 and pSTB-CEN4′ used in topology assays are drawn schematically. Recombination by the R recombinase within pSTB-CEN4 would resolve it into the pSTB plasmid plus the CEN4 circle. (Lower) The experimental regimen for obtaining cells for analyses of plasmid topology is schematically indicated. Raffinose was replaced by galactose in G1-arrested cells to induce expression of the R recombinase or the Rep proteins or both, as required by individual assays. Cells were normally released from G1 at 26 °C; they were released at 37 °C to inactivate Ndc10 in the ndc10-1 strain. The predominant fraction of cells was in metaphase, large budded with a single nucleus near the bud neck, at the time of harvest.

Fig. 2.

Topological distributions of a single-copy STB reporter plasmid when STB is maintained functional or nonfunctional. The pSTB-CEN4 reporter plasmid (Fig. 1) was resolved into pSTB and CEN4 circle in G1, and their topologies were analyzed in the ensuing metaphase. After electrophoresis in a 1.5% agarose gel (0.3 μg/mL chloroquine), the pSTB (A) and CEN4 circle (C) bands were revealed by Southern analysis. The topological distributions in A and C are plotted in B and D, respectively. The “0” topoisomer, because of potential overlap with the nicked circle (N), was omitted in deriving their centers (vertical lines).

Fig. 3.

Topologies of the pSTB plasmid in single copy state in G1 arrested or nocodazole-treated cells. (A–D) The single-copy pSTB and CEN4 circle were generated in G1 by recombination (see Fig. 2). Cells were released from G1 in galactose with the addition of nocodazole (Noc) or without nocodazole as the control (Con). Topology analyses were performed in G1 arrested cells (lane 2) or G2/M cells (Con, lane 1; Noc, lane 3).

Fig. 4.

Individual contributions of STB and CEN to the topology of a reporter plasmid harboring both loci. (A and B) The indicated strains harboring the pSTB-CEN4’ reporter plasmid (Fig. 1) were arrested in G1 at 26 °C, conditioned with galactose, and released in galactose at 26 °C or 37 °C. Plasmid topologies were assayed at 2 h after release.

Fig. 5.

Change in DNA topology between functional and nonfunctional STB. A ΔLk of ∼|2| because of the transition of STB between its functional (Lk_f) and nonfunctional (Lk_nf) states may be accommodated in one of at least three ways (A–C). (A) The right-handed DNA wrap around a Cse4-containing nucleosome (red) switches to the standard left-handed wrap around a histone H3-containing nucleosome (green). (B) A nucleosome-free, functional STB switches to a nonfunctional state occupied by two histone H3-containing nucleosomes. (C) Loss of two Cse4-containing nucleosomes, each with a positive-DNA writhe, from a functional STB generates a nucleosome-free, nonfunctional STB. These models assume that the standard twist of B-form DNA is not significantly altered when it is occupied by histone H3- or Cse4-containing nucleosomes.