Investigating Maintenance Of The Yeast 2-Micron Family Of Plasmids

Abstract

The 2µm plasmid of Saccharomyces cerevisiae is the best-studied member of a family of multi-copy plasmids found only in Saccharomycetaceae budding yeast. All members of the family are small circular double-stranded DNAs that share structural and functional similarities but lack significant nucleotide sequence identity. These plasmids use yeast replication and segregation machineries for their own maintenance but do not seem to benefit or pose a detriment to the host. For the 2µm plasmid, association of the plasmid-encoded Rep1 and Rep2 proteins with the cis-acting STB partitioning locus has been shown to be required for equal partitioning of plasmid copies at host cell division. Other members of the family, the 2µm-like plasmids, remain mainly uncharacterized. The goal of this research was to experimentally analyze 2µm-like plasmids pSR1 and pSB3 from Zygosaccharomyces rouxii and pKW1 from Lachancea waltii to gain a better understanding of the components and features required for plasmid maintenance, as well as to investigate the contribution of the 2µm Rep proteins to the plasmid partitioning mechanism. Plasmid inheritance assays were used to identify the 2µm-like pSB3 plasmid partitioning locus (PAR) and show that the pSB3 plasmid could efficiently partition in a heterologous S. cerevisiae host. Despite this, the partitioning loci of pSR1, pSB3 and pKW1 could not functionally substitute for 2µm STB and increase the inheritance of a 2µm-based plasmid in S. cerevisiae. These results suggest that partitioning locus recognition by the plasmid proteins is plasmid-specific and sequence differences between the loci might determine the specificity of the cis-acting sequence. Consistent with this, the pSB3 partitioning proteins Rep1 and C recognized the pSB3 PAR sequence in vivo but could not associate with the 2µm STB DNA sequence. The pSR1 and pSB3 partitioning proteins associated with their partners in vivo, with pSB3 Rep1 and C interacting through their amino-terminal regions, as previously observed for 2µm Rep1 and Rep2. Additionally, the C-terminal domain of pSB3 Rep1 was required for the efficient association of the pSB3 C protein with the pSB3 PAR locus and plasmid gene promoters. Taken together, these results suggest that the mechanism of plasmid partitioning between pSB3 and 2µm plasmids is conserved as well as the transcriptional regulation of plasmid genes by the partitioning proteins. Artificial tethering of proteins to a non-partitioning plasmid was used to characterize a novel Rep1-independent role of 2µm Rep2 in 2µm plasmid partitioning and to show that motifs that enable this function can now be assessed by mutational substitutions. Furthermore, the C-terminal 65 amino acids of 2µm Rep2, required for this Rep1-independent function, could be functionally substituted by the corresponding C-terminal region of the pKW1 C partitioning protein. This research represents the first experimental analysis of partitioning proteins encoded by other members of the 2µm-like family of plasmids and has provided insight into a contribution of 2µm Rep2 to plasmid partitioning that is independent of its interactions with the Rep1 partitioning protein. Additionally, results obtained in this research suggest that all members of the family function in the same way which may reflect their evolution from a common ancestor. Furthermore, molecular tools developed in this study will now allow further investigation of the partitioning mechanism by which the 2µm-like plasmid family is stably maintained.