Replicating plasmids are highly unstable in yeast, because they are retained in mother cells. The 2 mu circle plasmid overcomes this maternal inheritance bias by using a partitioning system that involves the plasmid encoded proteins Rep1p and Rep2p, and the cis-acting locus STB. It is thus widely exploited as a cloning vehicle in yeast. However, little is known about the cellular or molecular mechanisms by which effective partitioning is achieved, and models of both free diffusion and plasmid localisation have been proposed. Here we show that Rep1p and Rep2p proteins interact to form homo- and hetero-complexes in vitro. In vivo, Rep1p and Rep2p are shown to be nuclear proteins, exhibiting sub-nuclear concentration in distinct foci. The number of foci appears constant regardless of plasmid copy number and cell ploidy level. Before cell division, the number of foci increases, and we observe approximately equal allocation of foci to mother and daughter cell nuclei. We show that whereas Rep2p expressed alone is found exclusively in the nucleus, Rep1p requires the presence of Rep2p for effective nuclear localisation. High levels of 2 mu plasmid induce a multiple-budded elongated cell phenotype, which we show can be phenocopied by overexpression of both REP1 and REP2 together but not alone. Taken together, these results suggest that Rep1p and Rep2p interact in vivo, and occupy defined nuclear sites that are allocated to both mother and daughter nuclei during division. We propose a model for 2 mum plasmid partitioning based on these results, involving the association of plasmid DNA with specific, segregated subnuclear sites.

• PMID: 9625741
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Scott-Drew S, Murray JA

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