This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The successful transmission of the 2 m circle plasmid from mother cell to daughter cell relies on the ability to overcome an intrinsic maternal inheritance bias. This is dependent on the plasmid proteins Rep1 and Rep2, the plasmid DNA locus STB, and other factors encoded within the yeast chromosomes. We have previously shown that Rsc2, a member of the RSC chromatin remodeling complex, is essential for 2 m plasmid stability (Wong et al., 2002). Recently, this has been attributed to Rsc2 s role in recruiting the cohesin complex to the STB locus, thus, enabling the 2 m plasmid to utilise the chromosomal segregation machinery (Huang et al., 2004). Rsc2 is 45% identical and shares the same domain organisation as Rsc1. Rsc1 is thought to be present in an alternate form of the RSC complex(Cairns et al., 1999), and is not required for 2 m plasmid stability. Additionally, overexpression of Rsc1 cannot suppress the rsc2 mutant s plasmid maintenance defect. We are interested in understanding how Rsc2-containing complexes mediate 2 m plasmid stability, what enables them to recruit cohesin. To this end, we propose a 2 hybrid screen with domains of Rsc1 and Rsc2 that have been implicated in protein-protein interactions. With the discovery of unique Rsc2 interactors, we hope to further detail the link between the Rsc2 complex and cohesin. We also propose a 2-hybrid screen using the 2 m plasmid-encoded Rep1 and Rep2 proteins as baits. The high throughput YRC system is particularly appropriate since 2 m plasmid is found at copy numbers around 100 in most wild type yeast strains and 2-hybrid libraries commercially available would lead to a prohibitive background of plasmid protein interactions. We believe that such a screen would identify important novel components allowing a fuller characterisation of the 2 m segregation system.
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