Contributions Of DNA Sequence Elements, Plasmid-Encoded Proteins And Host Proteins To Maintenance Of The Yeast 2-Micron Plasmid

Abstract

The 2-micron plasmid is found at high copy in the nuclei of most strains of the budding yeast Saccharomyces cerevisiae. Despite conferring no selective advantage to its host, the plasmid is able to persist due to mechanisms that ensure equal partitioning of plasmid copies at cell division and amplification of plasmid copy number in the rare event of unequal partitioning. Partitioning requires the plasmid-encoded Rep1 and Rep2 proteins and a repeated DNA sequence at the plasmid STB locus. Plasmid amplification is catalyzed by plasmid-encoded Flp recombinase, and regulated by Rep protein-mediated repression of the FLP gene; Raf, the fourth plasmid protein, relieves this repression. The goals of this study were to identify features of the Rep proteins and plasmid sequences required for their function, and to examine the role of Raf and host proteins in ensuring plasmid maintenance.

Experimental and bioinformatics analyses were used to identify residues in Rep2 required for its association with Rep1 and Rep2. Rep2 mutants that had lost either or both associations were used to show that Rep2 plasmid partitioning function was retained provided one of these associations was intact and the Raf protein was present. This, along with additional analyses, suggests that Raf might be a Rep2 homolog.

To gain insight into sequences required for STB function, synthetic STB sequences were generated and used to determine that two identical direct repeats of a 63-bp stretch of STB are sufficient to confer partitioning function. Mutation of either of two TGCA sequences, a TGCA-adjacent T-tract, or a CGCG sequence in both repeat copies impaired partitioning function, while mutation of either TGCA sequence impaired Rep protein association with STB. Mutational analysis of plasmid gene promoters showed that TGCA sequences were required for Rep protein-mediated repression, suggesting that TGCA is a Rep protein recognition motif.

Finally, genome-wide screens identified candidate host factors that might contribute to functional chromatin arrangement at STB, or to the toxicity associated with Rep protein overexpression.

These findings serve to further refine the model of 2-micron plasmid maintenance, thus improving our understanding of how this extrachromosomal element persists in its eukaryotic host.