Both of the Saccharomyces cerevisiae 2m circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino-or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2m-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxyterminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2m circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.Most strains of the budding yeast Saccharomyces cerevisiae contain an endogenous plasmid, the 2m circle. This 6,318-bp double-stranded circular DNA plasmid is located in the nucleus at approximately 60 copies per haploid cell and replicates autonomously from, but synchronously with, the chromosomal DNA (for a review, see reference 9). The 2m circle confers no phenotype or selective advantage on the host yeast; indeed, 2m plasmid-bearing ([cir ϩ ]) cells grow 1% more slowly than isogenic plasmid-free ([cir 0 ]) cells (10). Despite this disadvantage, the 2m plasmid displays a high level of mitotic stability. This stability results from the presence of a plasmid-encoded copy number amplification system and a partition mechanism which together ensure that the rates of plasmid loss in mitosis and meiosis are very low (4,10,16,18). Partitioning of the 2m plasmid requires two proteins encoded by the plasmid genes REP1 and REP2 and a cis-acting 2m locus termed STB (16,18). The role of these three components has been examined in a variety of studies involving mainly deletion or insertion analysis of 2m-derived plasmids (17,18,23). In the absence of any one of these three components, the 2m plasmid displays a strong maternal bias in inheritance; most plasmids are retained in the mother cell (22). The 2m circle partition system overcomes this bias by an as yet unknown mechanism.The Rep1 and Rep2 proteins mediate 2m plasmid segregation, but thei...
