Copy number and partition of the Saccharomyces cerevisiae 2 micron plasmid controlled by transcription regulators.

Abstract

The 2,m plasmid of Saccharomyces cerevisiae is maintained by the action of plasmid-encoded gene products that control copy number and promote equipartition of plasmid copies at cell division. We show that the REP) and REP2 plasmid-encoded gene products are master regulators that act in concert to autoregulate the level of their own transcripts and to regulate transcript levels of the FLP gene that promotes plasmid copy amplification. REP) and REP2 are also shown to repress transcription at REP3, the cis-acting site essential for plasmid equipartitioning. We propose a model in which REP3 acts by dislodging transcription apparatuses that otherwise cause plasmid molecules to adhere to the mother nucleus and segregate asymmetrically. On the basis of their ability to generate specific chromatin structures, we also propose that the REP] and REP2 gene products interact with different specific sequences found iterated in the 2ILm plasmid.The 2,m plasmid is a 6.3-kilobase (kb) multicopy circular DNA found in most strains of S. cerevisiae (2,3,12,30). Because the plasmid confers no selective advantage to its host, it must rely on an efficient maintenance system. The rate at which cells lacking 2,um (ciro cells) are generated, approximately 10-4 to 10-5 per cell per generation (13), indicates an aggregate stability comparable with that of individual chromosomes (16). The chromosomally encoded nuclear replication apparatus contributes to plasmid maintenance. Mutations or inhibitors that block chromosomal replication similarly affect the 2,m plasmid (26). Like chromosomes, each of the 50 to 100 copies of the 2ixm plasmid replicates once during S phase (46). Once replicated, however, plasmid and chromosomal DNAs appear to be partitioned within the dividing nucleus by distinct mechanisms. Whereas the partition of chromosomes is mediated by centromeric sequences (10), the 2,um plasmid relies on an apparently unrelated cis-acting element, REP3. As defined by its ability to enhance the stability of chimeric derivatives of the 2,m plasmid, REP3 function requires at least two of five 62-base-pair (bp) tandemly repeated sequences (21,22). This locus appears to correspond to the STB locus (24) for which sequences flanking the 62-bp repeats were also found necessary for stabilization. Recent work suggests that flanking sequences may enhance REP3 function by preventing transcription through the direct repeats (32).The stabilization of the 2,um plasmid by REP3 requires two trans-acting plasmid genes, REP] and REP2 (5,21,24,43 (46). Moreover, the REP system is able to stabilize plasmids in the absence of FLP-catalyzed recombination. It therefore seems that the REP3-mediated stabilization reflects a more equal distribution of plasmid copies at division rather than correction of an initially biased distribution.In a previous study (43) In this study, we have used two approaches to document the negative regulation of FLP and other plasmid gene transcript levels by REP gene products. We first describe increases in transcript levels that res...

Functional Domains of Yeast Plasmid-Encoded Rep Proteins

“…The FLP gene encodes a site-specific recombinase required for plasmid amplification, and the control of Flp expression by Rep proteins has been proposed as the mechanism by which 2m plasmid copy number is regulated (8, 37). Rep protein-mediated transcriptional repression, like Rep protein segregation function, requires the concerted action of both Rep1 and Rep2 (25,31,35). Taken together, these data suggest that Rep1 and Rep2 may function as part of a complex.…”

“…Experiments designed to reconstitute efficient 2m segregation by expressing different amounts of Rep1 and Rep2 in the cell have shown that the relative levels of the two proteins are important for their partitioning function (5, 6). In addition to having a role in plasmid partitioning, the Rep proteins repress transcription of the 2m plasmid FLP gene (25,27,31,35). The FLP gene encodes a site-specific recombinase required for plasmid amplification, and the control of Flp expression by Rep proteins has been proposed as the mechanism by which 2m plasmid copy number is regulated (8, 37).…”

Site-specific recombination of the circular 2 microns-like plasmid pKD1 requires integrity of the recombinase gene A and of the partitioning genes B and C

“…In conclusion, these results demonstrate that unlike the situation in the 2,um system, where the FLP enzyme alone is sufficient for site-specific recombination and the partitioning genes REPI and REP2 act only as transcription regulators (11,13,14), in pKD1 the partitioning genes B and C are also directly involved in plasmid recombination. The possible roles in transcription regulation of genes B and C of pKD1 still remain to be investigated, but the absence of the fourth open reading frame suggests that the mechanism of transcription control which is at work is different from the one in the 2,um circle.…”

Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance

“…5A). These transcripts encode proteins required for copy number control and segregation of the plasmid, and defects in their functions result in decreased 2m plasmid levels in mutant strains (Jayaram et al 1985;Murray et al 1987;Veit and Fangman 1988). Indeed, measurement of 2m DNA levels in our test strains shows a large decrease in the air1-D rrp6-D strain (Fig.…”