Yeast plasmid segregation: investigation of host/plasmid interactions

The 2-micron plasmid is a multi-copy circular DNA found in the nucleus of most strains of the bakers yeast, Saccharomyces cerevisiae. The 2-micron plasmid has been called the perfect "selfish" DNA; presence of the plasmid does not adversely affect the host but nor does it confer a benefit. The plasmid ensures its own maintenance by borrowing host machinery for its replication and segregation. Equal partitioning of plasmid copies during host cell division requires association of plasmid proteins, Rep1 and Rep2, with a repeated sequence at the plasmid STB locus. Association of the Rep proteins recruits host proteins that help establish a functional partitioning complex at STB. The Rep protein/STB complex allows the plasmid to hitchhike on segregating chromosomes or some component of the chromosome segregation apparatus but the mechanism remains to be established. One hypothesis proposes that the partitioning sequences of yeast chromosomes may have been acquired from an ancestral 2-micron plasmid. In this study, biochemical, molecular genetics, and cell biology approaches will be used to identify and investigate host proteins borrowed by the 2-micron plasmid that enable its inheritance. RNA expression promoted by the STB sequence is being assessed to determine if this has functional significance that might represent a feature shared with chromosomal partitioning sequences. DNA protection and protein-DNA association assays will be used to investigate the effect of absence of host proteins identified in our studies on organization of the STB chromatin and RNA expression directed by STB. Our studies will help identify host proteins that contribute to specific aspects of chromosome biology and reveal new functions for others. We are also using molecular genetic approaches to investigate the sequences required at STB for partitioning competence and a comparative genomics approach in which partitioning proteins and sequences of 2-micron-related plasmids in other species of budding yeast will be studied to further our understanding of the biology and evolution of these selfish DNAs. This project will determine how the 2-micron plasmid interacts with other components of the nucleus to ensure its efficient segregation. Understanding how the yeast plasmid exploits features of chromosome segregation may reveal how other genetic elements, such as viruses are maintained and provide insight into how partitioning function may have has arisen during evolution.