Mol Gen Genet (1992) 236:8-16 © Springer-Verlag 1992

Differential repair and recombination of psoralen damaged plasmid DNA in Saccharomyces cerevisiae Eun-Kyoung Han and Wilma A. Saffran Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, NY 11367, USA Received December 27, 1991 / Accepted July 1, 1992

Summary. Psoralen photoreaction with D N A produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair. Yeast replicating plasmids, carrying the HIS3, TRPI, and URA3 genes, were photoreacted with psoralen in vitro and transfected into Saccharomyces eerevisiae cells. Repair was assayed as the relative transformation efficiency. A recombination-deficient rad52 strain was the least efficient in the repair of psoralen-damaged plasraids; excision repair-deficient radl 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 radl 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 radl 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. Key words: Interstrand D N A crosslinks - Gene conversion - Plasmid integration - rad mutants - Photoreaction

Introduction D N A photoreaction with psoralen produces interstrand crosslinks, in a two-photon reaction (Hearst et al. 1984). Monoadducts to pyrimidines are initially formed at the 4',5' or 3,4 positions of psoralen; the 4',5' monoadducts Correspondence to: W. Saffran

can react further to generate crosslinks. The interstrand crosslinks are cytotoxic lesions which require the interaction of different D N A repair pathways for removal. In the yeast Saccharomyces cerevisiae both the excision and recombinational repair pathways are required for psoralen crosslink repair (Averbeck and Moustacchi 1975; Henriques and Moustacchi 1980). The excision repair, or RAD3, group of genes confers resistance to ultraviolet radiation on yeast cells, and mediates the excision of pyrimidine dimers, bulky adducts and some forms of alkylated bases from D N A (Friedberg 1988). The recombinational repair, or RAD52, group confers resistance to ionizing radiation and is required for the repair of double strand breaks (Kunz and Haynes 1982). tad52 mutants are defective in meiotic recombination (Game et al. 1980; Prakash et al. 1980), mating type interconversion and several forms of mitotic recombination (Malone and Esposito 1980), including spontaneous and induced reciprocal exchange between chromosomes, gene conversion (Jackson and Fink 1981), and integration of gapped plamids (Orr-Weaver et al. 1981). Incision of psoralen-photoreacted DNA requires the RAD1, RAD2, RAD3, RAD4, and RADIO genes of the RAD3 group (Miller et al. 1982) and generates double strand breaks in cells treated with crosslinking psoralen derivatives (Jachymczyk et al. 1981; Magafia-Schwencke et al. 1982). Rejoining of the D N A breaks is dependent upon RAD51, a member of the RAD52 group. Crosslinks induce both gene conversions and reciprocal exchanges in diploid yeast cells (Averbeck et al. 1987). We have investigated the repair of specific genes in yeast cells by photoreacting plasmids with psoralen, and assaying plasmid survival and associated recombination. Plasmids are useful probes for repair processes, as in vitro modification allows targeting of lesions to specific genes without damaging essential genes or other molecules within cells. In a previous study we found that recombination of non-replicating plasmids was induced by psoralen damage; this recombination required RAD52 gene function and showed a partial dependence on RADI (Saffran et al. 1992). Here we use replicating

plasmids to measure psoralen damage repair, as well as the induction of a wider range of recombination products, in wild-type and repair-deficient yeast strains. Plasmid repair was found to depend on both RAD3 and RAD52 group genes. The repair efficiency was further found to vary with the conditions of transformant selection. Psoralen modification induced several forms of recombination, including non-crossover plasmid gene conversion, plasmid integration associated with gene conversion, and conversion of the chromosomal allele; the pattern of recombination events varied among the different genes examined. Materials and methods

Yeast strains and plasmids. W303 is M A T e Ieu2-3,112 trpl-1 ade2-1 ura3-1 canl-lO0 his3-11,15 (Rothstein 1983). H32 (MATc~ radl :.'LEU2 trpl-1 1eu2-3,112 ade21 ura3-1 canI-lO0 his3-11,15) was derived from W303 by gene disruption (Ronne and Rothstein 1988). WS5 ( M A T e rad52: : LEU2 trpl-I 1eu2-3,112 ade2-1 ura3-1 canI-lO0 his3-11,15) was derived from W303 by one-step disruption of the RAD52 gene. Yeast cells were transfected with a BamHI fragment of the RAD52 coding sequence, disrupted at the BglII site by the LEU2 gene (Schild et al. 1983); Leu + transformants were selected. WS1-4C (MATa rad3-2 trpl-1 ade2-i ura3-1 his3-ii,15) was constructed by crossing X36B-36 (MATa rad3-2 ade2-1), obtained from the Yeast Genetic Stock Center, with W303. The yeast shuttle plasmid YRp12 (Stinchcomb et al. 1980) carries the yeast URA3 and TRP1-ARS sequences inserted into pBR322. Y R p H U T was constructed by inserting the HIS3 gene, on a 1.8 kb BamHI fragment, into the BamHI site of YRp12 (Struhl 1985). Plasmids were propagated in Escherichia coli strain DH5 and were isolated by alkaline lysis, followed by CsC1 gradient purification (Maniatis et al. 1982).

0.5 ~tg plasmid DNA. The spheroplasts were suspended in 0.5 ml SOS medium and aliquots were plated onto S D - h i s , S D - u r a and S D - t r p media. The plates were incubated at 30° C for 5 days, then scored for transformation. The 100% relative transformation level for each omission medium was determined as the average number of colonies produced by duplicate spheroplast samples transfected with undamaged Y R p H U T DNA. This number varied with the spheroplast preparation, but was in the range of 5000 to 10000 colonies per gg plasmid D N A in the RAD, radl and rad3 strains, and 1000 to 3000 colonies per gg in the rad52 strain. The relative transformation levels of the psoralen-reacted plasmid samples were calculated as the ratio of the number of colonies transformed by damaged D N A to colonies transformed by undamaged D N A in the same spheroplast preparation. Each experiment was repeated at least twice. For genetic analysis, colonies were replica plated to each of the omission media and scored after 2 days at 30° C. For determination of stability of the traits, colonies were replica plated to YPD and grown for 1 day at 30 ° C, three times in succession, then replicated to each of the omission media.

Southern analysis. Yeast genomic D N A was prepared from 10 ml YPD cultures according to Sherman et al. (1986), digested with restriction endonucleases and run on 0.8% agarose gels in TAE (40 mM TRIS-acetate, 1 mM EDTA pH 8.0) buffer. D N A was transferred to Gene Screen Plus membranes (DuPont) by alkaline blotring. Probes were prepared by nick translation of D N A with biotin dUTP, using a nick translation kit (GibcoBRL, Gaithersburg, Md.) and hybridized to the membranes according to the manufacturer's directions. The filters were visualized with the BRL Blue Gene system with alkaline phosphatase-conjugated streptavidin. Results

Plasmid DNA reactions. [3H]4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was obtained from HRI, Inc. (Emeryville, Calif.). D N A in TE buffer (10 mM TRISHC1, 1 mM EDTA pH 8.0) at a concentration of 50 to 100 gM in base pairs, was incubated in the dark with [3H]AMT (0.3 Ci/mmol), at concentrations of 0-5 gM, for 30 min. The samples were irradiated with 350 nm light in a Rayonet photoreactor (Southern New England Ultraviolet Co., Hamden, Conn.) for 10 min. Unreacted AMT was removed from the plasmids by ethanol precipitation and resuspension in 0.3 M sodium acetate, followed by reprecipitation with ethanol. The D N A was resuspended in TE buffer at a concentration of 100 to 200 gM. The ratio of psoralen adducts to plasmid molecules was calculated from the amount of bound psoralen, as measured by scintillation counting, and the concentration of D N A (Saffran et al. 1992). Yeast transformation. Yeast culture media were prepared according to Sherman et al. (1986). Yeast spheroplasts were transfected by the method of Beggs (1978) with

Repair of psoralen-damaged plasmid DNA The repair of psoralen-damaged plasmid DNA was assayed by measuring the transformation efficiency of photoreacted plasmids relative to an undamaged control. The plasmid Y R p H U T is a yeast-Escherichia coli shuttle vector which carries the HIS3, URA3 and TRPI genes cloned into pBR322 (Fig. 1); the ARS1 sequence associated with TRP1 allows extrachromosomal replication of the plasmid within yeast cells. Y R p H U T was photoreacted with the water-soluble psoralen derivative [3H]-aminomethyltrimethyl-psoralen (AMT) in vitro, and the number of adducts per plasmid molecule was measured. Yeast spheroplasts were transfected with the reacted plasmids and transformants were selected by plating onto histidine, uracil or tryptophan omission media. The results are presented in Fig. 2. Transformation efficiencies were similar for the undamaged controls in all three media; however, in the repairproficient RAD strain we saw small but reproducible

10

without adding plasmid DNA. These minus DNA controls had 1-2 Trp ÷ colonies per 107 cells plated, but no Urn + or His + colonies, indicating that trpl-i does undergo a higher level of reversion than urn3-1 or his311,15. However, after correction for this background there was still an excess of Trp ÷ colonies over Urn ÷ and His + colonies. An alternative explanation is that transfection with damaged plasmid increases reversion by inducing untargeted mutagenesis. Transfection with psoralen-reacted bacteriophage lambda DNA, which has no homology to yeast, produced no increase in Trp ÷ or Urn + colonies. Similarly, introduction of damaged samples of the nonreplicating plasmid pUCtS-HIS3 induced His ÷, but not Trp ÷ or Urn + colonies (Saffran et al. 1991). Untargeted mutagenesis by introduction of a damaged replicating plasmid was studied with pJK21 ; this plasmid contains the HIS3 and URA3 genes, as well as the ARS1 replication origin, but lacks TRP1. Transfection of psoralenphotoreacted pJK21 did not induce Trp + colonies (data not shown); this indicates that general mutagenesis is not induced by the introduction of psoralen-damaged DNA. The relative transformation efficiency of damaged YRpHUT DNA was also measured in strains deficient in the excision repair genes RAD1 and RAD3 or the recombinational repair gene RAD52 (Fig. 2). These strains had higher background levels of reversion to Trp ÷ than the RAD cells, in agreement with previous reports of enhanced mutagenesis in repair-deficient strains ~Kunz et al. ~1990). The transformation results

BstXl

Ap

Pvu~ ----q

YRpHUT

Sinai Apal

BamHI

Fig. 1. Structures of the yeast repIicating plasmid Y R p H U T . Solid line, pBR322 sequences; boxes, yeast genes

differences in the numbers of transformed colonies at high levels of psoralen modification. There were consistently more Trp ÷ than Urn ÷ or His ÷ transformants, suggesting that the level of repair depends upon the genetic marker being measured. A possible explanation for the larger number of Trp ÷ colonies is a higher reversion level of the trpt-t gene of the host cell, rather than repair of the damaged TRP1 gene on the plasmid. The level of reversion was measured in samples which had undergone mock transfections,

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20 40 60 80 100120 20 40 60 8,0 1 0 0 1 2 0 2 0 40 60 BO 100120 Adducts/plasmid

Fig. 2. Relative transformation frequencies of psoralen photoreacted Y R p H U T . Yeast spheroplasts were transfected with Y R p H U T D N A and plated onto tryptophan (filled circles), uracil Oqlled squares), and histidine OCilled triangles) omission media. Left panel, RAD and rad52; center panel, radl; right panel, rad3. The data points are the average of duplicate platings of single samples at each level of damage, except for the samples without psoralen modification, for which two independent transformations were performed. Note that, except in the case of rad52 hosts, transformation efficiences are always highest on trp omission medium. The 100%

relative transformation level for each curve was calculated as the average number of colonies produced by the duplicate undamaged plasmid samples. For the experiments shown these levels were as follows. In the RAD strain: His + 685, 547; Urn + 780, 843; Trp + 885, 897. In the radl strain: His + 679, 871; Urn + 762, 958; Trp + 423, 676. In the rad3 strain: His + 996, 1162; Urn + 1277, 1534; Trp + 1522, 1809. In the tad52 strain: His + 77, 96; Urn + 125, 128; Trp ÷ 84, 126. The relative transformation efficiencies to Trp ÷ have been corrected by subtracting background levels of trpi-1 reversion

11 Table 1. Enhanced transformation to Trp + by psoralen photoreacted plasmids. Comparison of yeast strains Strain

Na

D lo (Trp +)/Dlo (His + or U r a +) Ratio b

RAD radl rad3 rad52

6 7 3 3

1.25+_0.13 1.67_ 0.37 1.28 +_0.05 1.03 +_0.06

of 1.0 is expected if the relative rates of transformation by damaged plasmids are identical in the different selective media, but this ratio is seen only in the recombination-deficient rad52 strain. The calculated ratios for the recombination-proficient strains were all greater than 1.0, with the radl cells showing the greatest enhancement in transformation in the tryptophan omission medium. Thus, the RAD52 gene plays roles in both the repair of psoralen damage in plasmid DNA and in the generation of differential repair.

" N u m b e r of independent experiments b Dlo, Psoralen damage dose at 10% relative transformation

Analysis of transformants

were therefore corrected by subtracting the background levels of Trp + colonies, as measured in the minus DNA controls. Reversion to Ura + or His + was not seen in mock-transfected controls. The radl and rad3 strains had lower relative transformation efficiencies with damaged plasmid than repair-proficient yeast, while the relative transformation was further decreased in the rad52 cells. There were also strain variations in the excess of Trp + transformants over Ura + and His + transformants. The ratios of the Dlo values for Trp + to the Dlo values for Ura + or His + transformation are reported in Table 1. The absolute values of the survival parameters varied as much as two-fold between different preparations of photoreaeted plasmid DNA, perhaps because of variability in the relative amounts of crosslinks and monoadducts. However, the ratios of these parameters in the different transformant selection conditions, within individual dose-response curves, were reproducible. A ratio

The transformants were analyzed to determine phenotypic changes in strains harboring repaired plasmids. YRpHUT has no centromere sequences and is therefore unstable in the absence of selection. Thus, after extended growth on complete medium the majority of cells have lost the plasmid and no longer grow on histidine, tryptophan or uracil omission media; YRpHUT transformants are phenotypically unstable His+Ura+Trp +. The plasmid traits may, however, become stable by transfer to the chromosome, through plasmid integration or gene conversion. Gene conversion between the plasmid and chromosomal alleles can produce colonies which have lost one or more of the plasmid gene functions. Plasmid conversion by one of the mutant chromosomal alleles will produce colonies which have lost one of the gene functions tested. For example, conversion of the plasmid HIS3 allele by the chromosomal his3 allele will produce a HisUra+Trp + colony which is singly auxotrophic for histidine.

Table 2. Y R p H U T transformants with altered phenotypes Strain

Psoralen

H i s - U r a + Trp +

His ÷ U r a - Trp +

His ÷ U r a + Trp -

Total tested

A. Singly auxotrophic transformants RAD radl rad3

+ b

0.08 1.11 (13.9) c

0.04

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

Psoralen photoreaction with DNA produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair. Yeast...
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