Repair of plasmid DNA treated with 8-methoxypsoralen and long-wave UV light (lambda=365 nm) in wild type and mutant rad2 cells of Saccharomyces cerevisiae

The method of repeated irradiation has been used to study excision of 8-MOP monoadducts from plasmid and chromosomal DNA in cells of wild type and rad2 mutant of Saccharomyces cerevisiae. The measurement of kinetics of monoadduct removal from chromosomal DNA in intact and competent yeast cells showed that monoadducts were excised in both types of cells with normal repair, but this process was blocked in intact and competent cells of the rad2 mutant. The survival of pYF91 plasmid treated in vitro with 8-MOP plus near UV-light has been studied in the cells of the wild type and in incision-defective rad2 mutant by the measurement of cell transformation frequency. Episomic pYF91 plasmid used in these experiments contained the yeast nuclear LEU2 gene, a portion of 2 mkm DNA and DNA of bacterial plasmid pBR322 with resistance to ampicillin. The pYF91 plasmid was treated with 8-MOP plus near UV-light in vitro, then unbound 8-MOP was removed by dialysis. This DNA was used for transformation. The transformed yeast cells were irradiated repeatedly. The quantitative alteration of the yield of transformants, depending on the time of keeping these yeast cells in complete liquid medium at 30 degrees C, prior to repeated irradiation, allowed to measure the kinetics of monoadduct excision from plasmid DNA. It was shown that monoadducts were removed equally effectively from plasmid DNA introduced into cells of the wild type and rad2 mutant. Possibly, the repair system of both these strains provides excision of monoadducts from plasmid DNA, but this process is blocked in the rad2 mutant, relatively to monoadduct excision from chromosomal DNA.     

Transformation of yeast spheroplasts without cell fusion

The efficiency of genetic transformation of Saccharomyces cerevisiae spheroplasts has been increased 10- to 100-fold over previously published procedures. Optimal transformation frequencies for single-stranded and double-stranded replicating plasmids are 2 X 10(7) and 5 X 10(6) transformants/microgram, respectively. At saturating DNA concentrations, 12 and 3%, respectively, of the viable spheroplasts contain plasmid DNA. The percentage of transformants that have undergone nuclear fusion varies from 0.1 to 3%, indicating that fusion is not required for the uptake of DNA by yeast spheroplasts.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

Repair of UV-irradiated plasmid DNA in a Saccharomyces cerevisiae rad3 mutant deficient in excision-repair of pyrimidine dimers

Summary The repair of UV-irradiated DNA of plasmid pBB29 was studied in an incision-defective rad3-2 strain of Saccharomyces cerevisiae and in a uvrA6 strain of Escherichia coli by the measurement of cell transformation. Plasmid pBB29 used in these experiments contained as markers the DNA of nuclear yeast gene LEU-2 and DNA of the bacterial plasmid pBR327 with resistance to Tet and Amp enabling simultaneous screening of transformant cells in both microorganisms.We found that the yeast rad3-2 mutant, deficient in incision of UV-induced pyrimidine dimers in nuclear DNA, was fully capable of repairing such lessions in plasmid DNA. The repair efficiency was comparable to that of the wild-type cells. The E. coli uvrA6 mutant, deficient in a specific nuclease for pyrimidine dimer excision from chromosomal DNA, was unable to repair UV-damaged plasmid DNA. The difference in repair capacity between the uvrA6 mutant strain and the wild-type strain was of several thousand-fold.It seems that the rad3 mutation, which confers deficiency in the DNA excision-repair system in yeast, is limited only to the nuclear DNA.     

Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers

The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency. Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair. In E. coli the repair of UV-irradiated plasmid DNA is carried out only by the excision-repair system dependent on uvr genes. Treatment of UV-irradiated plasmid DNA with UV endonuclease from Micrococcus luteus greatly enhances the efficiency of transformation of E. coli uvr- mutants. Similar treatment with cell-free extracts of yeast rad1 mutant or wild-type strains as well as with nuclease BaL31, despite their ability for preferential cutting of UV damaged DNA, showed no influence on cell transformation.     

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.     

Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

The replication of double-stranded plasmids containing a single N-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae. The strains used were proficient or deficient for the activity of DNA polymerase ζ (REV3 and rev3Δ, respectively) in a mismatch and nucleotide excision repair-defective background (msh2Δ rad10Δ). The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication. To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, ³²P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3Δ strains, the two strands of an unmodified heteroduplex plasmid were replicated in ~80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair. However, in the presence of the AAF adduct, TLS occurred in only 8% of the REV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in the REV3 strain was abolished in the rev3Δ mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.     

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.     

Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective in the RAD1, RAD2, RAD4, and RAD14 loci was not affected by transformation with these plasmids. The 4.5-kb fragment was subcloned into the integrating yeast vector YIp5, and the resultant plasmid was used to transform the rad3-1 mutant to UV resistance. Both genetic and physical studies showed that this plasmid integrated by homologous recombination into the rad3 site uniquely. We conclude from these studies that the cloned DNA that transforms the rad3-1 mutant to UV resistance contains the yeast chromosomal RAD3 gene. The 4.5-kb fragment was mapped by restriction analysis, and studies on some of the subclones generated from this fragment indicate that the RAD3 gene is at least 1.5 kb in size.     

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.     

Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation.

In the yeast Saccharomyces cerevisiae, a double-strand chromosome break created by the HO endonuclease is frequently repaired in mitotically growing cells by recombination between flanking homologous regions, producing a deletion. We showed that single-stranded regions were formed on both sides of the double-strand break prior to the formation of the product. The kinetics of the single-stranded DNA were monitored in strains with the recombination-deficient mutations rad52 and rad50 as well as in the wild-type strain. In rad50 mutants, single-stranded DNA was generated at a slower rate than in the wild type, whereas rad52 mutants generated single-stranded DNA at a faster rate. Product formation was largely blocked in the rad52 mutant. In the rad50 rad52 double mutant, the effects were superimposed in that the exonucleolytic activity was slowed but product formation was blocked. rad50 appears to act before or at the same stage as rad52. We constructed strains containing two ura3 segments on one side of the HO cut site and one ura3 region on the other side to characterize how flanking repeats find each other. Deletions formed preterentially between the homologous regions closest to the double-strand break. By varying the size of the middle ura3 segment, we determined that recombination initiated by a double-strand break requires a minimum homologous length between 63 and 89 bp. In these competition experiments, the frequency of recombination was dependent on the length of homology in an approximately linear manner.     

High frequency excision of Ty elements during transformation of yeast.

Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated-linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time.     

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.     

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.     

Isolation of a non-tumor-inducing mutant of the Ti plasmid of Agrobacterium tumefaciens strain B6.

A nonpathogenic mutant of Agrobacterium tumefaciens strain B6 was isolated and its properties compared with the parental strain in an effort to localize the mutation. Both B6 and its mutant (B6-95) had similar colony color and morphology, were ketolactose positive, utilized octopine, and contained plasmid DNA. Kinetic analysis of DNA reannealing showed that total DNA homology and plasmid DNA homology between B6 and B6-95 was at least 90%. The length of both plasmids was found to be 58 micrometer. Plasmid DNA from both B6 and the mutant was digested with endonucleases and the fragments separated by agarose gel electrophoresis. In all cases the pattern for B6 was identical with that of B6(-95). The Ti plasmid from B6 and the mutant was transferred to an avirulent, plasmidless strain of A. tumefaciens by in vitro conjugation and transformation. All of the B6 transconjugants and transformants were virulent, whereas all of the mutant transconjugants and transformants were avirulent. Electrophoretic patterns of endonuclease-digested plasmid DNA from transformants were identical to those of plasmid DNA from B6. Therefore, we conclude that the virulence mutation lies on the Ti plasmid.     

A system for analysis of yeast mutants deficient in the genetic recombination process]

A system of molecular-genetic methods intended for analysis of yeast Rec-mutants was developed. A number of plasmids containing noncomplementing mutations in the ADE2 gene and different selective markers were constructed. The system was based upon the phenomenon of interplasmid intragenic recombination during transformation and mitotic division. Transformation of yeast cells with the plasmid containing two long right repeats allowed to estimate the efficiency of intraplasmid crossing-over. The system also allows to study plasmid-chromosomal interaction. To check up the system proposed, a well known rad50 and rad52 mutants were used. The rad52-1 mutation was found sharply decreased the levels both intra- and interplasmid recombination events. On the other hand, rad50-1 mutation has not influences or stimulates the processes. Thus, the system proposed efficiently distinguishes the mutants deficient in different stages of recombinational process.     

Isolation of recipient yeast strains for the stable reproduction of 2-micron DNA vectors

Most Leu- clones of yeast transformants (cir0, pJDB219) can stabilize the replication of 2 micron-vectors with REP3, the stability obtained being comparable to the one for the standard cir0 strain. One of the Leu- clones was used to isolate a plasmid with Rep 1.2 functions ("Rep-helper plasmid"). The plasmid was shown to carry a partially active LEU2 gene by transforming both E. coli and S. cerevisiae to Leu+ phenotype. A restriction analysis performed demonstrated that the Rep-helper plasmid has lost approximately 1.9 kb compared to the parent pJDB219, deletion and rearrangement having taken place at the bacterial and 2 mem components boundary. The Rep-helper plasmid carrying host strains allows to quantify the REP3 function on different 2 microns vectors. Some but not all cir+ stabilized vectors show greater stability in Rep-helper strains compared to the standard cir0 ones. Manipulating the Rep-helper plasmid level, by selecting for Leu+ phenotype, stabilized REP3 +/- plasmid p3030, but mostly destabilizes REP3+ plasmid YEp13HIS3.     

Error-free RAD52 pathway and error-prone REV3 pathway determines spontaneous mutagenesis in Saccharomyces cerevisiae

Using the CAN1 gene in haploid cells or heterozygous diploid cells, we characterized the effects of mutations in the RAD52 and REV3 genes of Saccharomyces cerevisiae in spontaneous mutagenesis. The mutation rate was 5-fold higher in the haploid rad52 strain and 2.5-fold lower in rev3 than in the wild-type strain. The rate in the rad52 rev3 strain was as low as in the wild-type strain, indicating the rad52 mutator phenotype to be dependent on REV3. Sequencing indicated that G:C-->T:A and G:C-->C:G transversions increased in the rad52 strain and decreased in the rev3 and rad52 rev3 strains, suggesting a role for REV3 in transversion mutagenesis. In diploid rev3 cells, frequencies of can1Delta::LEU2/can1Delta::LEU2 from CAN1/can1Delta::LEU2 due to recombination were increased over the wild-type level. Overall, in the absence of RAD52, REV3-dependent base-substitutions increased, while in the absence of REV3, RAD52-dependent recombination events increased. We further found that the rad52 mutant had an increased rate of chromosome loss and the rad52 rev3 double mutant had an enhanced chromosome loss mutator phenotype. Taken together, our study indicates that the error-free RAD52 pathway and error-prone REV3 pathway for rescuing replication fork arrest determine spontaneous mutagenesis, recombination, and genome instability.