Maintenance of the 2 microns circle plasmid in populations of Saccharomyces cerevisiae.

The 2 microns circle plasmid is maintained at high frequencies in populations of yeast cells. To find out how the plasmid is maintained, three forces were measured: the selective advantage or disadvantage conferred by 2 microns circles, the rate of generation of [Cir0] cells, and the rate of illegitimate transfer of 2 microns circles from cell to cell. It was found that under the conditions used, 2 microns circles confer a selective disadvantage of about 1%, that [Cir0] cells are generated at the rate of 7.6 x 10(-5) per [Cir+] cell per generation, and that illegitimate transfer of 2 microns circles occurs at a rate less than 10(-7) per recipient cell per generation. The most likely explanation of 2 microns circle maintenance is that the plasmid is sexually transmitted at such a rate that it spreads through populations despite selection against it.     

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.     

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.     

DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of the FLP protein and two inverted recombination sites on the plasmid. The minimal fully functional substrate for in-vitro recombination in this system consists of two FLP protein binding sites separated by an eight base-pair spacer sequence. We have used site-directed mutagenesis to generate every possible mutation (36 in all) within 11 base-pairs of one FLP protein binding site and the base-pair immediately flanking it. The base-pairs within the binding site can be separated into three classes on the basis of these results. Thirty of the 36 sequence changes, including all three at seven different positions (class I) produce a negligible or modest effect on FLP protein-promoted recombination. In particular, most transition mutations are well-tolerated in this system. In only one case do all three possible mutations produce large effects (class II). At three positions, clustered near the site at which DNA is cleaved by FLP protein, one of the two possible transversions produces a large effect on recombination, while the other two changes produce modest effects (class III). For seven mutants for which FLP protein binding was measured, a direct correlation between decreases in recombination activity and in binding was observed. Positive effects on the reaction potential of mutant sites are observed when the other FLP binding site in a single recombination site is unaltered or when the second recombination site in a reaction is wild-type. This suggests a functional interaction between FLP binding sites both in cis and in trans. When two mutant recombination sites (each with 1 altered FLP binding site) are recombined, the relative orientation of the mutations (parallel or antiparallel) has no effect on the result. These results provide an extensive substrate catalog to complement future studies in this system.     

Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle.

The FLP protein of the Saccharomyces cerevisiae plasmid 2 microns circle catalyzes site-specific recombination between two repeated segments present on the plasmid. In this paper we present results of experiments we performed to define more precisely the features of the FLP recognition target site, which we propose to designate FRT, and to determine the actual recombination crossover point in vivo. We found that essential sequences for the recombination event are limited to an 8-base-pair core sequence and two 13-base-pair repeated units immediately flanking it. This is the region identified as the FLP binding site in vitro and at which FLP protein promotes specific single-strand cleavages (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski, Cell 40:795-803, 1985; J. F. Senecoff, R. C. Bruckner, and M. M. Cox, Proc. Natl. Acad. Sci. USA 82:7270-7274, 1985). Mutations within the core domain can be suppressed by the presence of the identical mutation in the chromatid with which it recombines. However, mutations outside the core are not similarly suppressed. We found that strand exchange during FLP recombination occurs most of the time within the core region, proceeding through a heteroduplex intermediate. Finally, we found that most FLP-mediated events are reciprocal exchanges and that FLP-catalyzed gene conversions occur at low frequency. The low level of gene conversion associated with FLP recombination suggests that it proceeds by a breakage-joining reaction and that the two events are concerted.     

FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction

The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction.     

Curing of the 2 mu DNA plasmid from Saccharomyces cerevisiae.

The 2 mu DNA plasmid is often eliminated from yeast cells when they are transformed with the 2 mu DNA-LEU2-pMB9 composite plasmid pJDB219. Since pJDB219 is subsequently lost with high frequency, derivatives lacking all 2 mu DNA can be prepared from any strain.     

Ligation activity of FLP recombinase. The strand ligation activity of a site-specific recombinase using an activated DNA substrate.

The FLP protein of the 2-microns plasmid of yeast belongs to the integrase family of site-specific recombinases whose members form a covalent bond between a conserved tyrosine of the recombinase and the 3'-phosphoryl group at the site of cleavage. We have made an activated DNA substrate and have shown that FLP can promote efficient strand ligation without forming a covalent intermediate with the DNA substrate. The strand ligation activity of FLP is independent of its ability to cleave DNA. Since site-specific recombinases are members of the larger class of topoisomerases, these findings may be generally applicable to other members of this class of enzymes.     

The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae

Summary The survival of plasmid YRp12 treated in vitro with ultraviolet- or -radiation, or with restriction endonucleases, has been used to investigate in vivo RAD gene activity in Saccharomyces cerevisiae. Yields of pyrmidine dimers or single and double strand breaks in plasmid DNA were assayed by physical methods. The biological effects of these damages were assayed by transformation of wild-type cells and rad mutants from each of the major groups of radiosensitive mutants. After UV-irradiation plasmid survival depended qualitatively on the same host functions that are needed for cellular survival. After -irradiation no such correspondence was found. Apart from a RAD52-dependent stimulation of transformation efficiency at low doses, other host repair functions had little effect. Stimulation of transformation corresponded with the production of double- but not single-strand breaks in plasmid sequences homologous with the yeast genome and may be linked with a transient increase in mitotic stability.More generally these data also show that transformation events using the LiCl protocol may entail the uptake of a very low number of plasmid molecules per cell over a 10-fold range of DNA concentrations.     

Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae. A role for arginine 191 in binding and ligation.

The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts. Using site-directed mutagenesis, we have made mutations in this region of the FLP gene. Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site. A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination. This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA. FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites. The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination. The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.     

The mechanism of loading of the FLP recombinase onto its DNA target sequence

The FLP recombinase interacts with its target sequence with the formation of three distinct DNA-protein complexes. The first complex leaves neither a DNase footprint nor is the DNA protected from methylation by dimethyl sulfate. We have found, however, that the FLP protein is bound predominantly to only one of the three 13 base-pair (bp) symmetry elements. This asymmetric loading of the FLP site seems to require the presence of an adjacent directly repeated 13 bp element. We speculate that this asymmetric filling of the target site may be accompanied by the unique order of cleavage and exchange of DNA strands.     

Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae.

Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae. Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time. This suggested that repair tracts were frequently shorter than 1 kilobase. The two insertion mismatches were repaired with different efficiencies. Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches. When two different plasmids containing single-insertion mismatches were transformed into S. cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.     

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.     

Differential repair and recombination of psoralen damaged plasmid DNA in Saccharomyces cerevisiae.

Summary Psoralen photoreaction with DNA produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair. Yeast replicating plasmids, carrying the HIS3, TRP1, and URA3 genes, were photoreacted with psoralen in vitro and transfected into Saccharomyces cerevisiae cells. Repair was assayed as the relative transformation efficiency. A recombination-deficient rad52 strain was the least efficient in the repair of psoralen-damaged plasmids; excision repair-deficient rad1 and rad3 strains had repair efficiencies intermediate between those of rad52 and RAD cells. The level of repair also depended on the conditions of transformant selection; repair was more efficient in medium lacking tryptophan than in medium from which either histidine or uracil was omitted. The plasmid repair differential between these selective media was greatest in rad1 cells, and depended on RAD52. Plasmid-chromosome recombination was stimulated by psoralen damage, and required RAD52 function. Chromosome to plasmid gene conversion was seen most frequently at the HIS3 locus. In RAD and rad3 cells, the majority of the conversions were associated with plasmid integration, while in rad1 cells most were non-crossover events. Plasmid to chromosome gene conversion was observed most frequently at the TRP1 locus, and was accompanied by plasmid loss.     

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.     

Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae

Summary We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae. The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid. The frequency of lithium transformation of Rad⁺ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene. This effect is due to recombinational repair of the plasmid DNA. Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA. The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14–18 h of incubating cells on selective medium.     

Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1

The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ～106 Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.     

Heterogeneity among the 2 microns plasmids in Saccharomyces cerevisiae: a new sequence for the REP1 gene

Some species of yeasts contain naturally-occurring circular DNA plasmids. The most studied of these plasmids is the 2 microns circle of Saccharomyces cerevisiae. Three variants of this plasmid, Scp1, Scp2 and Scp3, have been described according to their restriction maps [Cameron et al., Nucleic Acids Res. 4 (1977) 1429-1448; Livingston, Genetics 86 (1977) 73-84]. The entire nucleotide (nt) sequence of the Scp1 variant from strain A364A has been published [Hartley and Donelson, Nature 286 (1980) 860-864]. We report here the nt sequence of the 2 microns plasmid REP1 gene from S. cerevisiae strain SKQ2n. According to the restriction analysis, this plasmid is the Scp3 variant previously described. The only observed differences between the Scp1 and Scp3 variants were the loss of one EcoRI restriction site and an apparent deletion in Scp3. The nt sequence we report differs significantly from the previously published one for Scp1. The differences correspond to 128 (about 8.5%) substituted, deleted or additional nt of 1510 nt compared. These differences affect the coding region (8%) as well as the noncoding regions (9.7%). Regarding the putative encoded proteins, 38 (about 10%) amino acids (aa) are modified or deleted in our sequence and 11 are added. Most of these aa modifications are not randomly distributed but are concentrated in certain regions. These observations are indicative of important intraspecific evolution between the two 2 microns plasmid variants considered, as well as of conservative selection pressure on some domains of the REP1 protein.     

The in vivo replication origin of the yeast 2 microns plasmid

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.     

Accumulation of single strand interruptions within the yeast 2 microns DNA plasmid during replication in a DNA ligase mutant

Summary We have investigated the fate of the yeast 2 m DNA plasmid in strains with a temperature sensitive mutation of DNA ligase. At the restrictive temperature the plasmid DNA collects as an open circular form with single strand interruptions. Both alpha factor pheromone, which arrests cells before the start of S phase, and hydroxyurea, which blocks progression through S phase, prevent the appearance of the open circular form. Thus, interrupted plasmid DNA does not accumulate in the absence of DNA replication. On average the interrupted molecules contain four to five interruptions per newly replicated strand. Most of the interruptions are nicks (breaks in a single phosphate ester bond) rather than gaps (absence of one or more nucleotides in a strand) as judged by the in vitro conversion of the interrupted molecules into a covalently closed form by DNA ligase. Mapping of the position of the interruptions reveals no predominate sites.