Chapter 14 : The 2μm Plasmid of

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The first part of this chapter is devoted to the 2µm circle partitioning system, a critical component of the plasmid's strategy for stable maintenance in yeast populations. The second part deals with plasmid copy number control, special attention being paid to the Flp recombination system that is believed to trigger a DNA amplification process. The chapter compares the 2µm plasmid with 2µm-like plasmids found in yeast and dwells briefly on the degree and the significance of conservation of structure and function among them. Binary fluorescence tagging of two separate plasmids in the same cell by using cyan fluorescence protein (CFP)-Lac repressor/Lac operators in one case and yellow fluorescent protein (YFP)-Tet repressor/Tet operators in the other is also feasible. Chromatin immunoprecipitation assays revealed that the integral cohesin component Mcd1p associates specifically with the STB DNA in a Rep1p- and Rep2p-dependent manner. Rank and colleagues have characterized 2µm plasmids from several amphiploid industrial strains of and analyzed their sequence divergence with respect to plasmids from standard haploid laboratory strains. The Flp-FRT site-specific recombination system complements the partitioning system in the dual strategy by which stable high-copy maintenance of the 2μm plasmid is achieved. Plasmids lacking STB tend to dissociate from these sites and wander toward the nuclear periphery. These observations would be in line with the models for plasmid segregation considered in this chapter.

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 1

Structural and functional organization of the 2μm plasmid. (A) The double-stranded circular plasmid is shown in the standard dumbbell form in which it is normally represented. The parallel lines (the handle of the dumbbell) indicate the inverted repeats (IRs) of the plasmid. The open reading frames are highlighted, with the arrowheads pointing in the direction of their transcription. The cis-acting DNA elements in the plasmid are the replication origin (ORI), the partitioning locus (.STB), and the Flp recombination target sites (FRT). (B) The STB element, contained between the indicated PstI and sites, can be subdivided into two regions: proximal and distal with respect to STB-proximal contains the tandem array of five to six copies of a 62-bp consensus sequence and is central to plasmid partitioning. STB-distal is important in maintaining the “active configuration” of .STB-proximal, which is subject to context effects. Two plasmid transcripts (,650 nucleotides [nt], 700 nt) directed toward the OR/ are terminated within .STB-distal. A third transcript ,950 nt) runs in the opposite direction and traverses the STB-distal region. The shaded box within .STB-distal represents a “silencer sequence” that can suppress the activity of a promoter placed in its vicinity in an orientation-independent manner ( ). It is believed that the directional termination of transcription within .STB-distal is required for the functional integrity of the partitioning locus. (C) The site consists of three 13-bp Hp-binding elements, la, I′a, and I′b, whose orientations are denoted by the horizontal arrows. The elements la and I′a, together with the 8-bp spacer region included between them, constitute the sequences directly relevant to the recombination reaction (the minimal site). The points at which strand cleavage and exchange occur arc indicated by the vertical arrows.

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 2

Requirement of the Rep proteins and spindle integrity for plasmid compactness. In the Z-series confocal microscopy, 20 consecutive sections (each 0.25 μm in thickness) are scanned. Every alternate section is shown here. The top row shows the pattern of an STB-containing reporter plasmid in a [cir+] strain (Rep1 and Rep2 proteins derived from the native 2um circles). Note the increase in the width of the plasmid residence zone in the absence of the Rep proteins ([cir+]; bottom row) or the absence of an intact spindle even when the Rep proteins are present ([cir+] treated with nocodazole; middle row).

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 3

Association of the yeast cohesin complex with the 2u.m plasmid. The chromatin immunoprecipitations are done with antibodies to the cohesin component Mcd1p fused to the HA epitope. The lanes are arranged as the positive controls WCF. (whole cell extract), the experimental samples (ChIP), and the mock-precipitated negative controls (beads only). (A) Mcd1p associates with STB and a cohesin-binding site on chromosome V (lane 2) but not with an ARS sequence (lane 5). (B) Mcd1p- STB association in a [cir0] strain does not occur in the presence of Rep1p alone (lane 2) or Rep2p alone (lane 5) but requires the presence of both proteins (lane 8). (C) When integral cohesin components Smc1p and Smc3p are inactivated by T mutations, Med I p fails to bind to STB, as it does to a chromosomal binding site (compare lane 2 to lane 5 and lane 8 to lane 11).

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 4

Recruitment of the cohesin complex by the 2μm plasmid as a function of the cell cycle. Cells arrested in GI by a factor are released from pheromone arrest at time zero and followed by chromatin immunoprecipitation (using Mcd1p-directed antibodies), light microscopy (DIC), and FACS analysis. During each cell cycle, association of cohesin with the STB element occurs early in S phase and lasts until late G2/M. Note the nearly perfect synchrony between the chromosomes and the plasmid in cohesin association and dissociation.

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 5

Noncleavable Mcd1p blocks the separation of duplicated plasmid clusters. Small budded cells harboring a copy of the native MCD1 gene and one of the noncleavable version (MCD1-nc) under GAL promoter are transferred from dextrose to galactose at time zero. They are followed for 150 min by time-lapse fluorescence microscopy to monitor a tagged chromosome (top two rows), an STB reporter plasmid (central two rows), or an ARS plasmid (bottom two rows). Of the 10 cells examined in each case (and arrested at the large budded state), the fractions exhibiting one chromosomal dot versus two dots and one plasmid cluster versus two clusters are indicated.

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 6

Plausible models for cohesin-mediated 2μm plasmid segregation. (A) The plasmid cluster is bridged by cohesin to its sister cluster following or concomitant with duplication. The two clusters in turn are tethered to a pair of sister chromatids. The tethering agent is unlikely to be cohesin itself. Upon cleavage of Mcd1p, the two clusters ride with the chromosomes to opposite cell poles. (B) The duplicated plasmid clusters are held together by cohesin as in A. However, their migration to opposite cell poles following cohesin disassembly is independent of chromosomes.

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 7

A recombination-mediated amplification mechanism proposed by Futcher ( ). Bidirectional replication starting at the origin in a plasmid molecule (a) duplicates the proximal FRT site before the distal one (b). A Flp-mediated inversion (c) results in two replication forks oriented in the same direction (d). Movement of the two forks around the circular template amplifies copy number (e). A second recombination event (f) restores bidirectional fork movement (g). The products of replication are a template copy (i) and an amplified moiety containing multiple tandem copies of the plasmid (h). The tandem multimer can be resolved by Flp recombination into plasmid monomers (j, k). The diagram of the Futcher model shown here follows its representation by Broach and Volkcrt ( ).

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 8

Geometry, chemistry, and dynamics of the Flp recombination reaction. Two DNA substrates (L1RI and L2R2), each bound by two Flp monomers, are brought together in antiparallel orientation with respect to each other. I. and R refer to the left and right DNA arms, with the suffix 1 or 2 indicating substrate 1 or substrate 2, respectively. One active site is assembled on each DNA partner (the left arms, as diagrammed here). Strand cleavage and exchange (indicated by the small arrowheads) result in the formation of the Holliday intermediate H1. During isomerization, the DNA arms flex to produce the H2 geometry. Cleavage pockets are now assembled on the right arms. Strand cutting and exchange resolve H2 into the recombinants L1R2 and L2R1. In reality, the Holliday intermediate of recombination has a nearly square planar configuration. The difference in the angles included between a given DNA arm and its adjacent partners in HI and H2 (between R1 and LI and LI and R2, for example) is exaggerated here to highlight the isomerization step. In the first strand-exchange reaction, an active dimer is formed by Flp monomers bound to the same DNA molecule (two darkly shaded Flps in one case and the two lightly shaded Flps in the other). In the second strand-exchange step, an active dimer is formed between a darkly shaded Flp and a lightly shaded one. The long continuous arcs ending in small circles indicate the catalytically relevant dimer interactions. The corresponding short discontinuous arcs indicate inactive dimer interactions.

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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Figure 9

Positive and negative controls of gene expression in the 2μm plasmid. The schematic diagram depicting 2μm circle gene regulation is adapted from Som et al. ( ). The putative bipartite regulator Replp-Rep2p (R1-R2) negatively controls expression of the FLP (Flp), RAF1 (D), and REP1 (Rl). As a result, the level of the R1-R2 repressor is controlled as a function of the copy number, and at steady state, the amplification system is essentially turned off. The product of the RAF1 gene (D) antagonizes RI-R2, permitting rapid triggering of recombination-mediated amplification when plasmid copy number needs a boost. The REP2 locus appears to be free from repression by R1-R2. Aside from their role in controlling plasmid gene expression, the Rep1 and Rep2 proteins interact with the STB DNA to bring about equal segregation of the plasmid molecules at cell division.

Citation: Jayaram M, Yang X, Mehta S, Voziyanov Y, Velmurugan S. 2004. The 2μm Plasmid of , p 303-324. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch14
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