Partitioning of the 2-mu m circle plasmid of Saccharomyces cerevisiae: Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution

The efficient partitioning of the 2-mu m plasmid of Saccharomyces cerevisia e at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host en coded factors are likely to contribute to plasmid segregation. Direct obser vation of a 2-mu m-derived plasmid in live yeast cells indicates that the m ultiple plasmid copies are located in the nucleus, predominantly in cluster s with characteristic shapes. Comparison to a single-tagged chromosome or t o a yeast centromeric plasmid shows that the segregation kinetics of the 2- mu m plasmid and the chromosome are quite similar during the yeast cell cyc le. Immunofluorescence analysis reveals that the plasmid is colocalized wit h 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 part ial dispersion of the Rep proteins in the nucleus concomitant with a loosen ing in the association between plasmid molecules. In an ipl1-2 yeast strain , shifted to the nonpermissive temperature, the chromosomes and plasmid alm ost always missegregate in tandem. Our results suggest that, after DNA repl ication, plasmid distribution to the daughter cells occurs in the form of s pecific DNA-protein aggregates. They further indicate that the plasmid part itioning mechanism may exploit at least some of the components of the cellu lar machinery required for chromosomal segregation.