They accomplish this remarkable evolutionary stability by using two different strategies to ensure their own transmission: partitioning and amplification (Velmurugan et al., 2003; Jayaram et al., 2004). After DNA replication, partitioning ensures a high fidelity of segregation of plasmid molecules. This requires two encoded proteins, Rep1 and Rep2, which bind specifically to a cis-acting STB (for stability) locus and, together, mediate this plasmid segregation. In the absence of such an active segregation mechanism (i.e., if plasmid segregation was simply diffusion dependent), the rate of loss of 2-μm plasmids from daughter cells coupled with the fitness advantage of Cir^o cells would lead to the rapid clonal extinction of 2-μm plasmids. In the case of a decrease in copy number below a steady state value, plasmid amplification reestablishes this steady state by increasing rounds of replication. This amplification uses an elaborate series of recombination events mediated by the encoded Flp recombinase and two cis-acting FRT (Flp recombinase target) sites using a single DNA replication initiation event (Futcher, 1986; Volkert and Broach, 1986).

All of this circumstantial evidence had pointed to the 2-μm plasmid usurping the chromosome segregation machinery from the host yeast cell to ensure its own partitioning. Now, Hajra et al. (2006) add a critical piece to this puzzle by demonstrating that the 2-μm plasmid utilizes the yeast centromeric histone Cse4 (Stoler et al., 1995) to ensure correct segregation. Centromeric histones like Cse4 are exquisitely specific markers of centromeric chromatin in virtually all eukaryotes. The authors first show that Cse4 and 2-μm plasmids colocalize in chromosome spreads. Using chromatin immunoprecipitation followed by PCR, they show that Cse4 localizes specifically to the STB locus. Using high-salt extractions, they show that the STB locus of the 2-μm plasmid indeed assembles a Cse4-containing nucleosome, which protects the STB locus from restriction enzyme–mediated cleavage. They further demonstrate that STB-localized Cse4 is protected from proteolytic degradation in the same manner as CEN (yeast centromere)-localized Cse4 (Collins et al., 2004). Finally, they demonstrate that Cse4 is genetically required for the correct partitioning of the 2-μm plasmid. In the absence of wild-type Cse4, Rep2 does not localize to STB (although Rep1 does), RSC2 complex–mediated chromatin remodeling does not take place, and cohesin assembly at STB is blocked. Indeed, Hajra et al. (2006) argue that correct partitioning requires correctly remodeled STB chromatin containing a Cse4 nucleosome, which can nucleate Rep1–STB–Rep2 interactions. These results together imply that the STB locus effectively mimics CEN function.

However, there are important differences between 2-μm plasmid and yeast chromosome segregation. The 3′ CDEIII element of CEN sequences specifically recruits proteins of the CBF3 complex, including Ndc10 (Goh and Kilmartin, 1993; Jiang et al., 1993), which in turn helps recruits a Cse4 nucleosome, most likely at the CDEII element based on genetic data (Fig. 1 A; Keith and Fitzgerald-Hayes, 2000). Thus, Cse4's localization to CEN sequences is disrupted in an ndc10-1 mutant at nonpermissive temperatures, but its localization to STB loci is unaffected. Conversely, although Cse4 localization to STB loci requires Rep1 and Rep2 proteins (Fig. 1 B), the deletion of Rep1 or Rep2 has no effect on its CEN localization. This implies that 2-μm plasmids have invented a new means to recruit Cse4 to their STB loci, which is probably key to their longevity. Hajra et al. (2006) speculate that a complex containing Rep1 and Rep2 may help deposit (and perhaps maintain) Cse4 at STB. This further implies that although some components of both the 2-μm plasmid and yeast kinetochores are bound to be in common, others (e.g., Ndc10) are not. Thus, the 2-μm plasmid kinetochore may prove to be valuable in future studies of CEN-based kinetochores.

Centromere function is under extremely strong constraints in budding yeasts, and the general architecture of CEN sequences has been largely preserved over tens of millions of years (Fig. 2 A). In contrast, STB loci are highly variable in sequence and especially in length even within S. cerevisiae (Fig. 2 B; Rank et al., 1994b). Previous studies have strongly suggested that this is a result of antagonism between 2-μm plasmids and their host cells (Murray et al., 1988; Rank et al., 1994a), as the latter try to evolve genetic solutions to their 2-μm infestations. In addition, 2-μm plasmid alleles compete with each other for survival within yeast cells (Rank et al., 1994a). This means that Cse4 is forced to negotiate with both very slowly evolving (CEN) as well as extremely rapidly evolving (STB) centromeres, which is an interesting challenge for an essential histone.