Studies of the maintenance of the 2 micrometre circle of Saccharomyces cerevisiae.

The 2 mum plasmid of Saccharomyces cerevisiae is a multicopy, nuclear DNA plasmid. The stable maintenance of this plasmid is dependent on both host and plasmid-encoded functions. It has been proposed that the 2 mum encodes functions involved in both plasmid partitioning, and copy number amplification. Prior to this work it had been demonstrated that the plasmid's B, C and STB loci were required for efficient partitioning during haploid vegetative growth. However, it had not been determined whether the same partitioning functions were required for stable plasmid maintenance during alternative developmental pathways of the host cell. This study suggests that trans-acting functions are also required for stable plasmid inheritance during both diploid mitotic growth and sporulation. Although the maintenance systems are very efficient, native 2 mum plasmids do become unstable in the presence of 2 mum based plasmids. The cause of this plasmid incompatibility is unknown. It is possible that it is due to competition for limited resources. This study demonstrates that the native plasmid is incompatible with plasmids carrying the cis-regions of the 2 mum, and with an ARS -based plasmid carrying no 2 mum sequences. These results suggest that 2 mum incompatibility does not arise due to functions specific to the 2 mum, but may involve host replication functions. The 2 mum plasmid's site specific recombination system is thought to be involved in copy number amplification. The 2 mum plasmid's site specific recombination system has been shown to be involved in copy number amplification previously, but to date the importance of this process for plasmid maintenance during vegetative growth has not been established. This study demonstrates that FLP-mediated recombination is required for amplification, since a 2 mum plasmid, with a cis-acting mutation in the site specific recombination system, is unable to amplify. Moreover, there is a significant increase in the number of 2 mum free segregants, and a wide distribution in copy number in the mutant 2mum population. This suggests that the partitioning mechanism results in an unequal distribution of molecules at cell division, and that copy number amplification compensates for this.