Partitioning of the 2-μm Circle Plasmid of Saccharomyces cerevisiae

Abstract

The efficient partitioning of the 2-μm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-μm–derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-μm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.

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

“…Neither model requires cohesin for plasmid clustering per se, which is likely mediated through the Rep proteins in conjunction with associated host factors. Consistent with this notion, the clustered plasmid organization is not altered in G 1 -arrested cells even though they lack cohesin at STB (7,15).…”

“…The generally accepted amplification mechanism is a carefully timed Flp recombination event that converts a pair of bidirectional replication forks into unidirectional ones by DNA inversion (13). The dual rolling-circle replication generates a plasmid concatamer that may be resolved by Flp recombination or homologous recombination.Despite a copy number of Ϸ40-60 per cell, the 2-m plasmid exists as a single tight-knit cluster of foci and segregates as a cluster (14,15). This copy-number reduction, effectively to unity, makes an active partitioning mechanism imperative for stable propagation.…”

“…Despite a copy number of Ϸ40-60 per cell, the 2-m plasmid exists as a single tight-knit cluster of foci and segregates as a cluster (14,15). This copy-number reduction, effectively to unity, makes an active partitioning mechanism imperative for stable propagation.…”

“…Synchronized cell populations were obtained by ␣ factor-induced arrest in G 1 phase, followed by release as described in ref. 15. Nocodazole arrest and subsequent release are detailed in refs.…”

“…10 and 15. Fluorescence microscopy of reporter plasmids, indirect immunofluorescence assays for cytoskeletal structures, and ChIP followed protocols standardized in prior studies (4,7,15).…”