Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae.

We have examined the replication and segregation of the Saccharomyces cerevisiae 2 microns circle. The amplification of the plasmid at low copy numbers requires site-specific recombination between the 2 microns inverted repeat sequences catalyzed by the plasmid-encoded FLP gene. No other 2 microns gene products are required. The overexpression of FLP in a strain carrying endogenous 2 microns leads to uncontrolled plasmid replication, longer cell cycles, and cell death. Two different assays show that the level of Flp activity decreases with increasing 2 microns copy number. This regulation requires the products of the REP1 and REP2 genes. These gene products also act together to ensure that 2 microns molecules are randomly segregated between mother and daughter cells at cell division.     

Genetic control of plasmid recombination in the cotransformation of Saccharomyces cerevisiae: the role of RAD52 and FLP genes

In our earlier works we observed high frequency of recombination between two chimeric plasmids of different types, when they were introduced into yeast cells via cotransformation. Incapability of one of these plasmids to replicate autonomously in yeast cell is the necessary condition for such recombination. The high efficiency of this process point to the differences between interplasmid recombination and other types of yeast recombination. In this work, we studied the participation of two genes in the control of interplasmid exchanges. These are RAD52 responsible for normal processes of meiotic and mitotic recombination and highly specific gene FLP located on 2 mkm DNA which specifies site-specific recombination in the region of inverted sequences of this plasmid. The mutation rad52 in the recipient strain was shown to sharply decrease the efficiency of recombination between integrative and episome plasmids during cotransformation. The absence of FLP gene in the recipient strain (cirO) has no influence on this process.     

The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae codes for a site-specific recombinase ('FLP') that efficiently catalyses recombination across the plasmid's two 599 bp repeats both in vivo and in vitro. We have used the partially purified FLP protein to define the minimal duplex DNA sequence required for intra- and intermolecular recombination in vitro. Previous DNase footprinting experiments had shown that FLP protected 50 bp of DNA around the recombination site. We made BAL31 deletions and synthetic FLP sites to show that the minimal length of the site that was able to recombine with a wild-type site was 22 bp. The site consists of two 7 bp inverted repeats surrounding an 8 bp core region. We also showed that the deleted sites recombined with themselves and that one of three 13 bp repeated elements within the FLP target sequence was not necessary for efficient recombination in vitro. Mutants lacking this redundant 13 bp element required a lower amount of FLP recombinase to achieve maximal yield of recombination than the wild type site. Finally, we discuss the structure of the FLP site in relation to the proposed function of FLP recombination in copy number amplification of the 2-micron plasmid in vivo.     

Recombination within the yeast plasmid 2mu circle is site-specific

The multicopy yeast plasmid, 2mu circle, encodes a specialized recombination system. It contains two regions, each 599 bp in length, that are precise inverted repeats of each other and between which recombination occurs readily. In addition, this recombination requires the product of a 2mu circle gene, designated FLP. By examining the products of FLP-mediated recombination of plasmids containing single insertions within one of the repeated regions, we show that this recombination occurs only at a specific site within the repeat. This result was confirmed from analysis of the ability of plasmids containing various deletions within one of the repeated regions to serve as substrates for FLP-mediated recombination. These experiments limit the recombination site to a sequence of less than 65 bp. In addition, by mutational analysis of the recombination potential of a hybrid plasmid containing the entire 2mu circle genome, we have shown that FLP is only the 2mu circle gene necessary for this site-specific recombination. Finally, we describe a sensitive assay for recombination between the repeated sequences of 2mu circle; using it, we demonstrate that even in the absence of FLP gene product, recombination between the repeats occurs at a low but detectable level during meiosis.     

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level.

The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSM1 (5,416 base pairs), isolated from Zygosaccharomyces fermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2 microns DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSR1, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2 micron DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSR1 are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSR1 showed clear homology with that of open reading frame B of pSB2.     

Site-specific recombination promotes plasmid amplification in yeast

All stable, naturally occurring circular yeast DNA plasmids contain a pair of long, nontandem inverted repeats that undergo frequent reciprocal recombination. This yields two plasmid inversion isomers that exist in the cell in equal numbers. In the 2 mu circle plasmid of S. cerevisiae such inversion is catalyzed by a plasmid-encoded site-specific recombinase, FLP. We show that the site-specific recombination system of 2 mu circle enables the plasmid to increase its mean intracellular copy number in yeast cells growing under nonselective conditions. This apparently occurs by a FLP-induced transient shift in the mode of replication from theta to double rolling circle as initially proposed by Futcher. This capability may ensure stable maintenance of the plasmid by enabling it to correct downward deviations in copy number that result from imprecision of the plasmid-encoded partitioning system.     

DNA plasmid transmission in yeast is associated with specific sub-nuclear localisation during cell division

Circular plasmids in yeast carrying only an origin of DNA replication (ARS) exhibit maternal inheritance bias (MIB) and are poorly transmitted from mother to daughter cell during division. A variety of different sequences that overcome MIB have been described, including centromeric sequences (CEN), telomere-associated repeats, silencer sequences and a specific system encoded by the endogenous 2 micron circle plasmid requiring the cis-acting locus STB and the proteins Rep1 and Rep2. In each case, DNA segregation between mother and daughter cells is dependent on DNA-protein interactions. Using plasmids carrying multiple copies of a lac repressor binding sequence, we have localised DNA molecules in the yeast nucleus using a green fluorescent protein (GFP)-lac repressor fusion protein. We compared GFP localised plasmids carrying a centromere sequence with plasmids based on 2 micron circle carrying or lacking the STB sequences required for their segregation. We show that GFP localised plasmid carrying the complete STB locus co-localises with the plasmid proteins Rep1 and Rep2 to discrete chromatin sites. These sites are distinct from both the telomeres and from sites of cohesin binding. Deletion of the region of STB essential for the stability of the plasmid, leads to a loss of plasmid association with chromatin, relocalisation of plasmids towards the nuclear periphery, and a decrease in the Rep1 protein associated with the plasmid. We conclude that specific plasmid localisation is likely to be important in the overcoming of MIB in yeast.     

Copy number control by a yeast centromere

Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome. In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that normally drives the 2 mu plasmids to high copy number. The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern. Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number. Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.     

The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene

Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

The 2microm-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus.

The efficient partitioning of the 2microm plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cis-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir+ strain. Nuclear localization of GFP-Rep1p and GFP-Rep2p, though discernible, is less efficient in a cir(0) host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2microm circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir+ cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2microm circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.     

Purification of the FLP site-specific recombinase by affinity chromatography and re-examination of basic properties of the system.

The FLP protein, a site-specific recombinase encoded by the 2 micron plasmid of yeast, has been purified to near homogeneity from extracts of E. coli cells in which the protein has been expressed. The purification is a three column procedure, the final step employing affinity chromatography. The affinity ligand consists of a DNA polymer with multiple FLP protein binding sites arranged in tandem repeats. This protocol yields 2 mg of FLP protein which is 85% pure. The purified protein is highly active, stable for several months at -70 degrees C and free of detectable nucleases. The molecular weight and N-terminal sequence are identical to that predicted for the FLP protein by the DNA sequence of the gene. Purified FLP protein primarily, but not exclusively, promotes intramolecular recombination. Intermolecular recombination becomes the dominant reaction when E. coli extracts containing no FLP protein are added to the reaction mixture. These extracts are not specifically required for recombination, but demonstrate that some properties previously attributed to FLP protein can be assigned to contaminating proteins present in E. coli.     

Insertion of transposon Tn9 into the spinal (Escherichia coli-Saccharomyces cerevisiae) plasmids and the expression of the prokaryotic gene of chloramphenicol resistance in yeast cells

Transposon Tn9 carrying camr gene which controls resistance to chloramphenicol has been introduced in vivo (in cells of Escherichia coli) into two chimeric shuttle plasmids pYF91 and YEp13. These plasmids consist of the different parts of the E. coli plasmid pBR322, the yeast 2mkm DNA plasmid and the yeast LEU2 structural gene. The plasmidis able to autonomously replicate in both yeast and bacterial cells. A recipient yeast strain carrying cams and leu2 markers was constructed to study the functional expression of the prokaryotic camr gene in eukaryotic yeast cells. The chimeric plasmids pYF91::Tn9 and YEp13::Tn9 were introduced into the yeast and bacterial recipient strains by transformation. The camr LEU2 yeast transformants were isolated. They were genetically unstable when grown on non-selective medium and they simultaneously lost camr and LEU2 markers with a frequency of 10 to 30%. The E. coli transformants were genetically stable under nonselective conditions and they maintain all plasmid markers. The chimeric plasmid pYF91::Tn9 was isolated from the yeast transformants and reintroduced into the cams leuB bacterial strain by transformation. The camr LEUB transformants were obtained. All these data confirm the possibility of the expression of the prokaryotic camr gene in yeast cells and present evidence for introduction of transposon Tn9 into chimeric plasmids.     

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.     

Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences.

The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro. We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo. One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein. This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence. The second mutation lies within the 8-bp core region of the FLP target sequence. The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange. The mutation in the core region abolishes recombination with a wild-type site. However, recombination between two mutated sites is very efficient. This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.