Faithful segregation of the multicopy yeast plasmid through cohesin-mediated recognition of sisters

Abstract

The 2-microm yeast plasmid, a benign high-copy nuclear parasite, propagates itself with nearly the same fidelity as the chromosomes of its host. Equal plasmid segregation is absolutely dependent on the cohesin complex assembled at the plasmid partitioning locus STB. However, the mechanism of cohesin action in the context of multiple plasmid copies, resident within two separate clusters after DNA replication, is unknown. By using "single-copy" derivatives of the 2-microm plasmid, we demonstrate that recruitment of cohesin at STB during S phase indeed translates into cohesion between plasmid molecules. Through binary fluorescence tagging, we reveal that segregation of replicated plasmids occurs in a sister-to-sister fashion. Thus, cohesin serves the same fundamental purpose in plasmid and chromosome segregation.

Fig. 1.

CEN–STB containing single-copy derivatives of 2-μm circle reporter plasmids. (A) The plasmids pSG1 and pSG2 contain, in addition to the 2-μm circle replication origin and STB, the CEN3 sequence flanked by the GAL promoter and the CYC1 terminator; pSG1 harbors a [LacO]₂₅₆ array, and pSG2 harbors a [TetO] ₁₁₂ array. (B) Total RNA from [cir⁺] and [cir⁰] strains harboring pSG1 was isolated after transferring raffinose-grown log-phase cells to medium containing glucose or galactose for the indicated times. RT-PCR was performed by using primers specific to the CEN3, STB, and TRP1 sequences. A DNA sample isolated from the [cir⁰]/pSG1 strain before galactose induction was amplified with the same primers as those used in the RT-PCR assays to provide the reference bands.

Fig. 2.

Plamsid cohesion mediated through STB or CEN during metaphase in cycling cells. (A) The experimental regimen for cell cycle arrest and release is schematically outlined. (B) The four representative classes of cells scored in the assay are displayed. Classes I and II are preanaphase cells showing one and two fluorescent plasmid foci, respectively. Classes III and IV are postanaphase cells containing 1:1 and 2:0 patterns of plasmid segregation, respectively. (C and D) DNA replication was followed by FACS analysis. (E) The cell populations were assayed at the indicated times after release from G₁ arrest and binned into classes I–IV.

Fig. 3.

Plasmid cohesion in cse4-1 [cir⁺] and smc1-2 [cir⁺] cells at permissive and nonpermissive temperatures. (A) The protocol for assaying plasmid cohesion is schematically indicated. (B and C) The fractions of metaphase cells displaying one fluorescent dot (cohesion) or two fluorescent dots (lack of cohesion) were estimated in the cse4-1 and smc1-2 strains at 26°C and 37°C.

Fig. 4.

Spindle integrity in the establishment of plasmid cohesion. (A) The effect of spindle disassembly on plasmid cohesion was followed according to the outlined experimental procedure. (B and C) The frequencies of one fluorescent plasmid dot (cohesion) or two fluorescent plasmid dots (lack of cohesion) were estimated in metaphase cells during the cell cycle (− nocodazole) or in cells arrested at G₂/M (+ nocodazole).

Fig. 5.

Lack of plasmid cohesion in cells released from nocodazole arrest into nocodazole-free medium. (A) The experimental scheme for nocodazole-mediated arrest of cells at G₂/M and their subsequent release to resume the cell cycle in the absence of the drug is diagrammed schematically. (B) Plasmid cohesion was assayed at the indicated time points during recovery. In parallel, the association of the cohesin component Mcd1p (expressed from the native promoter as an HA-tagged version) with STB was followed by ChIP. (C) The restoration of the mitotic spindle was monitored in recovering cells. The spindle was visualized in fixed cells by indirect immunofluorescence by using primary antibodies against Tub1p followed by fluorescein-conjugated secondary antibodies. (D) The segregation patterns of a CEN plasmid (green) and a cohabiting CEN–STB plasmid (red) under Rep-STB control were assayed in postanaphase cells 75 min after release from nocodazole arrest.

Fig. 6.

A sister-to-sister binary counting mechanism for the segregation of the 2-μm plasmid. (A) The possible modes of cohesin-mediated plasmid counting and segregation are schematically diagrammed. Replication of the single-copy reporter plasmids (one tagged by green fluorescence and the other by red fluorescence) generates two green and two red plasmid molecules. A strict sister-to-sister counting mode will pair red with red and green with green (I). A random counting mechanism may follow the pairing scheme I or mediate red-to-green pairings as indicated by II and III. Segregation according to I will not generate daughter nuclei containing two red or two green plasmids. However, equal probabilities of I, II, and III segregation modes will result in 33% frequency of such nuclei. (B) The distribution and organization of duplicated plasmid molecules according to the global counting mechanism (Left) and the sister-to-sister counting mechanism (Right) are schematically represented. The cohesin bridge (yellow) that pairs plasmid molecules (red and green) defines the boundary between the sister clusters that separate from each other upon cohesin disassembly. (C) Plasmid segregation was assayed in postanaphase cells under conditions where the plasmid-borne CEN and STB were both active, only one of the two was active, and neither one was active. The observed patterns were divided into classes I–V. Class I, with each prescient daughter cell containing one red and one green plasmid, is the only class predicted by the sister-to-sister segregation mode. Note that, even with an active centromere, class I was <100%. Class V, with would-be daughter cells containing two red or two green plasmids, is excluded by sister-to-sister segregation; however, it is a significant entity (33%) according to the random pairing mode. Note the rarity of class V in the presence of an active CEN or STB. Classes II–IV signify 3:1 and 4:0 missegregation events.