The 2-μm plasmid encoded protein Raf1 regulates both stability and copy number of the plasmid by blocking the formation of the Rep1–Rep2 repressor complex

The 2-μm plasmid of the budding yeast Saccharomyces cerevisiae achieves a high chromosome-like stability with the help of four plasmid-encoded (Rep1, Rep2, Raf1 and Flp) and several host-encoded proteins. Rep1 and Rep2 and the DNA locus STB form the partitioning system ensuring equal segregation of the plasmid. The Flp recombinase and its target sites FRTs form the amplification system which is responsible for the steady state plasmid copy number. In this work we show that the absence of Raf1 can affect both the plasmid stability and the steady sate copy number. We also show that the Rep proteins do bind to the promoter regions of the 2-μm encoded genes, as predicted by earlier models and Raf1 indeed blocks the formation of the Rep1–Rep2 repressor complex not by blocking the transcription of the REP1 and REP2 genes but by physically associating with the Rep proteins and negating their interactions. This explains the role of Raf1 in both the partitioning and the amplification systems as the Rep1–Rep2 complex is believed to modulate both these systems. Based on this study, we have provided, from a systems biology perspective, a model for the mechanism of the 2-μm plasmid maintenance.