Use of a cis-acting mutation to study the role of FLP-mediated recombination in the maintenance of native yeast 2 micrometer plasmids.

The 2 micrometer plasmid encodes a mechanism that ensures the partitioning of the plasmid at cell division. Little is known about the detailed mechanism of this partitioning system; for example, is there equal or unequal distribution of the plasmid molecules at mitosis? The plasmid also encodes a site-specific recombination system that is thought to be involved in plasmid copy-number amplification, although to date there has been no direct evidence that the recombination process itself is important for maintenance. We have identified a natural 2 micrometer variant that has a cis-acting mutation in the FLP-mediated recombination system. We show that this plasmid is unable to amplify in vivo. Our results demonstrate that the average copy number per cell is not affected for the mutant but there is a large clonal variation. This is a direct demonstration that plasmid partitioning results in an unequal distribution of plasmids and that FLP-mediated amplification compensates for this and therefore has an important role in maintenance.