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 hostcell 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.CEN evolution | nucleosome topology | reversome T he 2-μm plasmid of Saccharomyces cerevisiae resides in the nucleus at 40 to 60 copies per cell, and propagates itself with nearly the same stability as chromosomes (1, 2). The plasmid is a benign selfish DNA element that seems to provide no advantage to its host, but poses, at its normal copy number, no serious disadvantage either. In haploid cells the plasmid is organized into a cluster of three to five foci, and segregates as a cluster (3). This effective reduction in copy number necessitates an active partitioning system, comprised of two plasmid-coded proteins, Rep1 and Rep2, and a cis-acting partitioning locus STB, to ensure equal or nearly equal plasmid segregation. A decline in plasmid copy number because of rare missegregation events is corrected via DNA amplification triggered by the Flp sitespecific recombination system harbored by the plasmid (4, 5). Positive and negative regulatory circuits implemented through the plasmid-coded Raf1 protein and the Rep proteins ensure quick amplification response without the danger of runaway increase in copy number (6-8).The Rep-STB system channels several host factors involved in chromosome segregation toward the execution of plasmid segregation. These factors include the mitotic spindle, the spindleassociated motor Kip1, the RSC2 chromatin-remodeling complex, the centromere-specific histone H3-variant Cse4 (CenH3), and the yeast cohesin complex (9-15). The de novo assembly of the plasmid-partitioning complex at STB during the G1-S window of each cell cycle (9,12,14,16) is reminiscent of the assembly of the kinetochore complex at centromeres. However, kinetochore components have not been detected at STB by ChIP (13).The assembly of the plasmid-partitioning complex culmin...

Hijacking of Host Calreticulin Is Required for the White Spot Syndrome Virus Replication Cycle

“…This is in line with our results showing that CRT and VP15 together interact with WSSV DNA and so may affect replication or DNA packaging. A common feature of most DNA-binding or histone-like proteins is the ability to form DNA supercoils (49). In this work, we showed that VP15 as well as CRT preferentially bind to supercoiled DNA as previously shown (11,48).…”

Tripartite organization of centromeric chromatin in budding yeast

“…Most important is the histone variant, CenH3 (CENP-A in mammals and Cse4 in yeast), which replaces histone H3 in centromeric nucleosomes and is essential for recruitment of the other structural components of the kinetochore (5). It has been previously shown that the Cse4 nucleosome wraps DNA in a right-handed orientation (6,7), consistent with in vivo observations of heterotypic tetrameric nucleosomes in Drosophila (8) and humans (9). However, several studies have shown that CenH3 nucleosomes are left-handed octamers in vitro (10)(11)(12)(13)(14).…”

CENPT bridges adjacent CENPA nucleosomes on young human α-satellite dimers

“…Insofar as budding yeast CENPA nucleosomes also wrap DNA with right-handed chirality in vivo (Furuyama and Henikoff 2009;Huang et al 2011;Díaz-Ingelmo et al 2015), it is tempting to speculate that CENPA and CENPT subcomplexes coordinate to wrap a right-handed inner kinetochore chromatin complex. As DNA overwinds when stretched (Gore et al 2006), a right-handed wrap would be expected to tighten up under anaphase tension to provide a solid foundation for chromosome segregation (Fig.…”