The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function

Abstract

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.

Anchoring of parasitic plasmids to inactive regions of eukaryotic chromosomes through nucleosome signal

“…The dimer Rep1/Rep2 has been shown to associate with the plasmid STB sequence to promote partitioning during cell division and both STB and REP1/REP2 are required for the plasmid stability in host cell (McQuaid et al, 2019; Mereshchuk et al, 2022). We therefore tested whether the distribution of contacts along the genome was dependent on these partners by generating Hi-C data of cells with a 2µ plasmid mutants lacking either REP1 or the STB loci (Kikuchi, 1983; McQuaid et al, 2019) ( Methods ).…”

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae

“…Chromosome integration of episomal plasmids contributed to improved growth and improved nerolidol/valencene production. In Hxk2p-depleted/ hxk2Δ background (Figures , , and D,F), proteomic data showed that Rep2p, a key factor for 2μ plasmid inherence, was not present in Hxk2p-depleted cells. However, the activities of the REP2 promoter (and the TEF1 promoter as a reference) and the levels of the Rep2-yEGFP fusion under the control of the TEF1 promoter were not dramatically impacted by HXK2 disruption (Supplementary Figure 12).…”

The selfish yeast plasmid exploits a SWI/SNF-type chromatin remodeling complex for hitchhiking on chromosomes and ensuring high-fidelity propagation

“…In principle then, an ARS-plasmid non-covalently linked to the RSC complex should be able to overcome mother bias and improve its equal segregation frequency. In order to test this possibility, we relied on a [repressor-operator]-based experimental design described previously for recruiting proteins to specific sequences on plasmids to follow their segregation (Kiermaier et al, 2009;Lacefield et al, 2009;Mereshchuk et al, 2022).…”