Mutations in a Partitioning Protein and Altered Chromatin Structure at the Partitioning Locus Prevent Cohesin Recruitment by the Saccharomyces cerevisiae Plasmid and Cause Plasmid Missegregation

Abstract

The 2m circle is a highly persistent "selfish" DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes. The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway. The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules equally to daughter cells. The DNAprotein and protein-protein interactions of the partitioning system, as well as the chromatin organization at STB, are important for cohesin recruitment. Rep1p variants that are incompetent in binding to Rep2p, STB, or both fail to assist the assembly of the cohesin complex at STB and are nonfunctional in plasmid maintenance. Preventing the cohesin-STB association without impeding Rep1p-Rep2p-STB interactions also causes plasmid missegregation. During the yeast cell cycle, the Rep1p and Rep2p proteins are expelled from STB during a short interval between the late G 1 and early S phases. This dissociation and reassociation event ensures that cohesin loading at STB is replication dependent and is coordinated with chromosomal cohesin recruitment. In an rsc2⌬ yeast strain lacking a specific chromatin remodeling complex and exhibiting a high degree of plasmid loss, neither Rep1p nor the cohesin complex can be recruited to STB. The phenotypes of the Rep1p mutations and of the rsc2⌬ mutant are consistent with the role of cohesin in plasmid partitioning being analogous to that in chromosome partitioning.The 2m circle of Saccharomyces cerevisiae is a high-copynumber selfish extrachromosomal DNA element that resides in the nucleus and propagates itself stably in the cell population (2, 29). The plasmid does not seem to confer any selective advantage to its host under normal laboratory growth conditions, nor does it pose any noticeable disadvantage as long as the copy number does not rise significantly above the steadystate value of approximately 60 per cell. The stability of the plasmid approaches that of the yeast chromosomes, with the loss rate being as low as 10 Ϫ4 to 10 Ϫ5 per cell per generation. The structural organization of the plasmid and its genetic potential are devoted to two goals: (i) efficient plasmid segregation during cell division and (ii) the maintenance of the plasmid copy number with only modest deviations from the mean.The presence of a typical yeast replication origin in its sequence permits each plasmid molecule to be replicated once per cell cycle (35). A stability system consisting of two plasmidencoded proteins (Rep1p and Rep2p) and a cis-acting partitioning locus (STB) mediates equal or nearly equal distribution of the replicated molecules into daughter cells. The direct observation of reporter plasmids tagged with fluorescence indicates that the plasmid molecules are organized into a tightknit cluster in the nucleus and segregate as a cluster. Hence, the copy number relevant ...

Misregulation of 2μm Circle Copy Number in a SUMO Pathway Mutant

“…These strains contained only the tagged version of 2m, with none of the original native plasmid (see Materials and Methods). While the plasmids expressing tagged Rep1 and Rep2 were not maintained as well as the Flp1-and Raf1-expressing plasmids (ϳ1% loss per generation for tagged Rep1 and Rep2 versus 0.3% for Flp1 and 0.08% for Raf1) (see Materials and Methods), these loss rates are an order of magnitude lower than those seen when the plasmid segregation system is inactive (34,42,44). This suggested that tagged Rep1 and Rep2 were at least partially active.…”

“…Sumoylation of the other sites in Flp1 was Siz dependent. Several yeast proteins involved in 2m partitioning have recently been shown to be sumoylated, including the chromatin remodeling complex subunit Rsc2 and the cohesin subunits Smc1 and Scc1/Mcd1 (27,41,42,44). It is not known to what extent sumoylation of these proteins is controlled by Siz1 and Siz2.…”

“…In the absence of these factors, 2m remains primarily in the mother cell. Rep1 and Rep2 bind STB and inter-act with the cellular cohesin complex, and these interactions are essential for accurate plasmid segregation (27,44). The other mechanism enables 2m to be amplified if its copy number drops.…”

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

“…STB function depends on its association with Cse4, presumably as a nucleosome component, in a Rep1-Rep2-assisted manner (12,24, and present study). Furthermore, G1 arrest and spindle disassembly, conditions that do not foster Cse4-STB association (12) but sustain Rep-STB association (3,9,14,16), decrease Lk by the anticipated amount ( Fig. 3 A and B).…”

“…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)(10)(11)(12)(13)(14)(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).…”