Micro Review Bending the rules: the 2μ plasmid of yeast

The replication of euckaryotic DNA is normally initiated at each origin only once per ceil cycle. Yet, In spite of this restriction, the 2μ plasmid of yeast has evolved an elegant mechanism which can allow it to rapidly amplify its copy number without initiating multiple rounds of replication. It achieves this by exploiting a plasmid-en coded site-specific recombination system in a way that is apparently unique to this plasmid. The 2μ plasmid has also evolved a mechanism that allows effective partition of itself between mother and daughter cells. Together these processes ensure the persistence of the 2μ plasmid within a population, even though retention of the plasmid Is of no advantage to the host cell and causes a slightly slower growth rate. The success of this survival strategy is illustrated by the near ubiquity of the 2μ. plasmid in both wild-type and laboratory strains of yeast.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae

The 2 micron circle is a small double stranded DNA plasmid that occurs at about 60 copies per cell in the nuclei of virtually all strains of Saccharomyces cerevisiae. The plasmid has no apparent phenotypic effect on host cells, and is the basis of many useful vectors for the transformation of yeast. Under certain circumstances, the plasmid is apparently able to replicate more than once per cell cycle; this over-replication allows the maintenance of the plasmid at high copy number. The plasmid has two inverted repeat sequences, and encodes a product that catalyses intra-molecular recombination between these two repeats. Models are proposed whereby recombination leads to copy number amplification. In particular, it is proposed that intra-molecular recombination during replication flips the orientation of one replication fork with respect to the other, so that both forks travel in the same direction around a circular monomer template, generating a large multimer from a monomer and a single initiation of replication.     

Partitioning of the 2-microm circle plasmid of Saccharomyces cerevisiae. Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution

The efficient partitioning of the 2-microm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-microm-derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-microm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.     

Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.     

AT-rich sequences from the arbuscular mycorrhizal fungus Gigaspora rosea exhibit ARS function in the yeast Saccharomyces cerevisiae

Autonomous replicating sequences are DNA elements that trigger DNA replication and are widely used in the development of episomal transformation vectors for fungi. In this paper, a genomic library from the mycorrhizal fungus Gigaspora rosea was constructed in the integrative plasmid YIp5 and screened in the budding yeast Saccharomyces cerevisiae for sequences that act as ARS and trigger plasmid replication. Two genetic elements (GrARS2, GrARS6) promoted high-rates of yeast transformation. Sequence analysis of these elements shows them to be AT-rich (72-80%) and to contain multiple near-matches to the yeast autonomous consensus sequences ACS and EACS. GrARS2 contained a putative miniature inverted-repeat transposable element (MITE) delimited by 28-bp terminal inverted repeats (TIRs). Disruption of this element and removal of one TIR increased plasmid stability several fold. The potential for palindromes to affect DNA replication is discussed.     

A new system for amplifying 2 μm plasmid copy number in Saccharomyces cerevisiae

The yeast 2 microns plasmid is found in the nucleus of almost all Saccharomyces cerevisiae strains. Its replication is very similar to that of chromosomal DNA. Although the plasmid does not encode essential genes it is stably maintained in the yeast population and exhibits only a small, though detectable, loss rate. This stability is achieved by a plasmid-encoded copy-number control system which ensures constant plasmid levels. For the investigation of 2 microns replication, a yeast strain that is absolutely dependent on this plasmid was constructed. This was achieved by disruption of the chromosomal CDC9 gene, coding for DNA ligase and providing this essential gene on a 2 microns-derived plasmid. This plasmid is absolutely stable under all growth conditions tested. Using the temperature-sensitive mutant allele cdc9-1 we have developed an artificial control system which allows one to change the copy number of 2 microns-derived plasmids solely by changing the incubation temperature.     

Origin plasticity during budding yeast DNA replication in vitro

The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre‐replicative complexes (pre‐RCs) license origins by loading Mcm2‐7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2‐7 DNA helicase. Budding yeast pre‐RCs have previously been reconstituted in vitro with purified proteins. Here, we show that reconstituted pre‐RCs support replication of plasmid DNA in yeast cell extracts in a reaction that exhibits hallmarks of cellular replication initiation. Plasmid replication in vitro results in the generation of covalently closed circular daughter molecules, indicating that the system recapitulates the initiation, elongation, and termination stages of DNA replication. Unexpectedly, yeast origin DNA is not strictly required for DNA replication in vitro, as heterologous DNA sequences could support replication of plasmid molecules. Our findings support the notion that epigenetic mechanisms are important for determining replication origin sites in budding yeast, highlighting mechanistic principles of replication origin specification that are common among eukaryotes.     

Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells.

The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined.     

Analysis of a 1.6-μm circular plasmid from the yeast Kluyveromyces drosophilarum: Structure and molecular dimorphism

A new plasmid has been found in the yeast Kluyveromyces drosophilarum. It is a double-stranded circular DNA, 1.6 micron in length (4.8 kilobase pairs). As in the case of Saccharomyces 2 mu circles, this plasmid occurs in two isomeric forms corresponding to the inversion of a segment between two 346-bp-long inverted repeats within the molecule. Each form has been separately cloned into bacterial plasmids. The new yeast plasmid, called pKD1, contains sequences that allow its replication in Saccharomyces cerevisiae.     

ARS replication during the yeast S phase

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.     

A Conserved DNA Damage Response Pathway Responsible for Coupling the Cell Division Cycle to the Circadian and Metabolic Cycles

The circadian clock drives endogenous oscillations of cellular and physiological processes with a periodicity of approximately 24 h. Progression of the cell division cycle (CDC) has been found to be coupled to the circadian clock, and it has been postulated that gating of the CDC by the circadian cycle may have evolved to protect DNA from the mutagenic effects of ultraviolet light. When grown under nutrient-limiting conditions in a chemostat, prototrophic strains of budding yeast, Saccharomyces cerevisiae, adopt a robust metabolic cycle of ultradian dimensions that temporally compartmentalizes essential cellular events. The CDC is gated by this yeast metabolic cycle (YMC), with DNA replication strictly segregated away from the oxidative phase when cells are actively respiring. Mutants impaired in such gating allow DNA replication to take place during the respiratory phase of the YMC and have been found to suffer significantly elevated rates of spontaneous mutation. Analogous to the circadian cycle, the YMC also employs the conserved DNA checkpoint kinase Rad53/Chk2 to facilitate coupling with the CDC. These studies highlight an evolutionarily conserved mechanism that seems to confine cell division to particular temporal windows to prevent DNA damage. We hypothesize that DNA damage itself might constitute a “zeitgeber”, or time giver, for both the circadian cycle and the metabolic cycle. We discuss these findings in the context of a unifying theme underlying the circadian and metabolic cycles, and explore the relevance of cell cycle gating to human diseases including cancer.     

Recombinant plasmids carrying multiple markers: isolation during yeast co-transformation

Cotransformants of yeast cells by two partially homologous plasmids, one of which is incapable of autonomous replication, has been used to construct multiply marked recombinant plasmids. Only simultaneous elimination of three yeast markers was registered when episomal plasmid, carrying Ade2 gene, and integrative plasmid, carrying yeast genes LEU2 and URA3, were cotransformed. Transformants, in which yeast genes LEU2, URA3 and HIS3 are linked, have been isolated by analogous technique. The genetic analysis has confirmed existence of plasmid cointegrates in the transformant cells, which carry three yeast genes, bacterial DNA fragment and 2 micrometers DNA fragment, coding for replicative functions. Recombination in the region of bacterial plasmid pBR322 might have resulted in formation of such plasmids. Plasmid recombination in cotransformants has been used to construct multiply marked circular chromosomes, having included yeast genes LEU2, URA3 and TRP1, centromere of the IV yeast chromosome and the sequence coding for their replication in yeast as well as in E. coli cells.     

Direct simulation of yeast 2-microns circle plasmid amplification.

The 2-microns circle is a plasmid found in most strains of the yeast Saccharomyces cerevisiae at approximately 60-100 copies per cell. The plasmid possesses the novel capacity for replicative amplification induced by site-specific recombination. To address the question of whether the recombination model is adequate to account for observed rates of 2-microns circle amplification, we developed a direct computational simulation of the amplification system. Results of this simulation show that theoretically at least six copies per plasmid can be produced in each generation, and that previously unanticipated replication intermediates contribute largely to this degree of amplification.     

The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response

Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid. Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication. Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid-borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess. We show that delayed cell division occurs by RepA-mediated induction of the SOS response and can be reversed by over-expression of the host DNA primase, DnaG. The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites. The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls--potentially pre-empting plasmid loss in cultures of dividing cells.     

Plasmid stability in budding yeast populations: Steady-state growth with selection pressure

Plasmid gene product accumulation in a cell population depends on the fraction of plasmid-containing cells and the distribution of single-cell plasmid content. These important population properties have been related to plasmid replication regulation and kinetics and to plasmid segregation rules at the single-cell level using population balance mathematical models. Budding yeast populations are considered in detail because of the practical potential of yeast host-vector systems and because of the model complications introduced by the asymmetric division pattern observed for Saccharomyces cerevisiae at all but the largest growth rates. Solutions are presented for several different reasonable models of plasmid replication and segregation. The results offer potential for identification of important qualitative features of yeast plasmid replication and of model parameter values from average and segregated experimental data on yeast populations.     

Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene.

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.