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,

1

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NAD o.1

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0.01

.

•\ 0.001

r

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I

I

I

I

I

I

i I

i

I

I

-, I

I

I

I

1

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I

i

I

i

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 <0.06 nd d

0.08 0.29 (3.6)

2400 1643

÷

0.12 0.51 (4.2)

< 0.06 0.58 (9.7)

< 0.06 0.26 (4.3)

1600 1242

-

0.50 1.34 (2.7)

0.08 0.16 (2.0)

0.45 0.35 (0.8)

2400 658

- ~

+ rad52

+

< 0.07 <0.47nd

< 0.07 <0.47 nd

< 0.07 <0.47 nd

1482 212

B. Singly prototrophic transformants RAD radl rad3

+

0.33 0.75 (2.3)

+

< 0.06 1.45 (24.2)

-

0.75 1.98 (2,6)

-

+ rad52

" b ¢ d

+

< 0.07 <0.47 nd

0.04 0.07 (1.8)

0.47 1.06 (2.3)

2400 1643

< 0.06 0.26 (4.3)

0.19 •.63 (8.6)

1600 •242

0.83 5.40 (6.5)

2400 658

0.04 0.90 (22.5) < 0.07 <0.47 nd

No psoralen reaction Psoralen adducts/plasmid in R A D , r a d l and rad3; 10 psoralen adducts/plasmid in r a d 5 2 Relative stimulation ( + p s o r a l e n / - psoralen) Not determined

< 0.07 <0.47 nd

1482 212

12 Table 3. Stability of singly auxotrophic transformants Phenotype

No. of stable transformants

His-Ura+Trp + His+Ura-Trp + His+Ura+Trp -

+

r~,dl

I

0.1

.... 0

,

,

,

I

40

,

,,

,

1

i

60

,

,

radi

rad3

29/41" (71)b 4/5 (80) 7/14 (50)

5/14 (36) 2/12 (17) 0/5 (0)

27/37 (73) 3/4 (75) 19/24 (79)

a Number of colonies with stable traits/total singly auxotrophic colonies tested b Percentage stable colonies

A I

20

RAD

I

,

80

,

,

I

100

,

,

~

120

I

B 2

3

4

I

2

Adducts/plosmid Fig. 3. Induction of histidine auxotrophs in YRpHUT transformants. Transformed colonies were tested for histidine, uracil and tryptophan prototrophy. The percentage of total His-Ura+Trp + transformants is plotted against psoralen adduct level. Open circles, radi transformants; closed circles, rad3 transformants

-3.7

-2.3 Chromosome conversion by one of the wild-type plasmid genes will produce colonies which have just one functioning gene on the chromosome. For example, conversion of the chromosomal allele to H I S 3 will produce a H i s + U r a - T r p - colony which is singly prototrophic for histidine. Furthermore, the His ÷ trait, being on the chromosome, is stable. If Y R p H U T is still present, the cells will be phenotypically H i s + U r a + T r p ÷, but only the trait present on the chromosome will be stable, e.g. stable His ÷, unstable U r a + T r p +. Transformants were tested for histidine, uracil and tryptophan prototrophy, as well as for the stability of each trait after growth on nonselective medium. Singly auxotrophic colonies

Psoralen damage induced singly auxotrophic Y R p H U T transformants in a dose-dependent manner (Table 2 and Fig. 3). The most frequent change was to histidine auxotrophy in the R A D and rad3 strains, and to uracil auxotrophy in the radl strain. The rad3 cells showed the highest levels of single auxotrophy after transformation with both unmodified and psoralen-damaged plasmids; both spontaneous and induced recombination were highest in this strain. The largest stimulations in recombination by psoralen damage were measured in the R A D strain for histidine auxotrophy and the radl strain for uracil and tryptophan auxotrophy. In the rad52 strain only one singly auxotrophic U r a - colony was found among the colonies transformed with undamaged plasmid, and none among those transformed with damaged plasmid. No H i s - or T r p - single auxotrophs were detected in this strain. Both stable and unstable single auxotrophs were detected (Table 3). In R A D and tad3 cells the majority of single auxotrophs, whether H i s - , U r a - , or T r p - , were stable for the other two traits. In contrast, most radl

-1.9

-1,4

Fig. 4A, B. Plasmid YRpHUT integration associated with gene conversion. A His Ura+Trp + cells with YRpHUT integrated at the HIS3 locus. Total cellular DNA was digested with EcoRI, and hybridized to a HIS3 probe. Lane 1, unstable His +Ura +Trp +, with extrachromosomal YRpHUT. The HIS3 probe hybridizes to a 7.3 kb fragment of the plasmid and a 10.0 kb fragment carrying the chromosomal HIS3 gene. Lanes 2 and 3, stable His-Ura +Trp +. YRpHUT integration into the HIS3 locus produces 13.5 and 4.5 kb bands. Lane 2 has a multiple integration, which produces an additional 7.3 kb band of plasmid sequences. Lane 4, parental cells without plasmid have the chromosomal 10.0 kb band only. B His+Ura+Trp - cells with YRpHUT integrated at the TRP1 locus. Cellular DNA was digested with SspI and hybridized to a TRP1 probe. Lane 1, stable His+Ura+Trp - cells; lane 2, parental cells single auxotrophs were unstable for the remaining traits. Stability of two plasmid genes implies chromosomal residence, probably achieved by plasmid integration associated with gene conversion. Thus, plasmid integration at the chromosomal his3 locus, accompanied by conversion of the plasmid H I S 3 alllele to the mutant chromosomal allele, should result in a stable H i s - U r a + T r p + phenotype. Plasmid integration was investigated by Southern blotting and hybridization with probes specific for each affected gene. Genomic D N A was prepared from stable H i s - U r a ÷ T r p + colonies, and digested with EcoRI; Southern blots were then hybridized to a H I S 3 probe. The results are presented in Fig. 4A. EcoRI digestion

13 of the intact chromosomal HIS3 gene produces a 10.0 kb fragment. YRpHUT integration disrupts this fragment and produces two new fragments, of 13.5 and 4.5 kb, that hybridize to the HIS3 probe. The integration of multiple copies of YRpHUT results in the appearance of an additional 7.3 kb band. Of 19 stable His-Ura ÷ Trp ÷ colonies tested, 10 were single integrations at the HIS3 locus, and 6 were multiple integrations. In the remaining three samples the chromosomal HIS3 gene was intact. When genomic DNA from these colonies was left undigested, a pBR322 probe hybridized to the high molecular weight chromosomal band, indicating that YRpHUT was chromosomally integrated, although at a site other than HIS3. Stable His+Ura+Trp - colonies were also examined for integration at the chromosomal trpl locus. Genomic DNA was digested with SspI and Southern blots were hybridized to a T R P I probe (Fig. 4 B). The intact chromosomal TRP1 gene produced a 2.1 kb SspI fragment; after integration of YRpHUT, bands of 1.8 and 9 kb result. Of 18 stable His+Ura+Trp - colonies tested, 15 had the 1.8 kb bands characteristic of plasmid integration at the TRP1 locus. Thus the majority of stable, singly auxotrophic colonies examined had plasmid integrated at the chromosomal locus homologous to the affected gene; this is consistent with a recombination event coupling gene conversion to reciprocal exchange.

10

-4D-

I

bD

I

o

03 -1-

0.1 0

~ ~ ~ I

~ ~ ~ I

~ ~ ~ I

~ ~ ~ I

20

40

60

80

,

~ ,

L ~ ~ t

1 O0

120

Adducts/plasmid Fig. 5. Induction of tryptophan single prototrophs by psoralen photoreaction of YRpHUT. The percentage of total YRpHUT transformants with a His-Ura- Trp ÷ phenotypeis plotted against psoralen modificationlevel. The number of His-Ura-Trp + colonies was corrected by subtracting the background level of trpl-i revertants in mock-transfectedcontrols prior to calculatingthe percent tryptophan prototrophs. Open circles, radI transformants; closed circles, rad3 transformants

Singly prototrophic colonies Prototrophy for only one of the three nutrients tested was found in colonies selected for each trait. H i s - U r a Trp ÷ colonies were the most frequent, followed by His+Ura-Trp - and, at a lower frequency, His-Ura ÷ Trp- colonies (Table 2). As discussed above, the trpl-1 allele exhibited measurable reversion; the data presented in Table 1 and Fig.5 have been corrected by subtraction of this background. Dose-response curves for the induction of His-Ura-Trp ÷ colonies are presented in Fig. 5, and for His+Ura-Trp - colonies in Fig. 6. The curves exhibit different dose dependencies in all strains tested; while the frequency of Trp ÷ cells is still increasing at the highest psoralen modification level tested, that of single His ÷ prototrophs reaches a maximum, then decreases. We found no induction above background of single prototrophs in the rad52 strain. Of the recombinationproficient strains, the highest frequencies of singly prototrophic colonies were found in the rad3 strain. The spontaneous frequency of single prototrophs, in colonies transfected by undamaged plasmids, was also elevated in this strain. Stimulation of single prototrophy by psoralen damage was greatest in the radl strain for histidine and tryptophan prototrophy, and in rad3 cells for uracil prototrophy. All of the singly prototrophic colonies were stable for the tested trait, indicating a chromosomal location of the allele. This may be produced by gene conversion of the chromosomal alleles to wild type, accompanied by loss of the other plasmid gene functions. Sixteen colo-

3 I

/

I

D + 217. 2

1

20

40

60

80

1 O0

120

Psoralens/plasmid Fig. 6. Induction of histidine single prototrophs by psoralen photoreaction of YRpHUT. The percentage of total YRpHUT transformants with a His+Ura-Trp - phenotypeis plotted against psoralen modificationlevel. Filled squares, RAD transformants;filled circles, radI transformants,filled triangles, rad3 transformants

hies each of singly prototrophic Trp ÷ and His ÷ colonies were tested by Southern analysis, using pBR322 as a hybridization probe. No hybridization was detected to either chromosome or plasmid-sized DNA, indicating that plasmid sequences were not present either as integrants or as extrachromosomal copies (data not shown). This result is consistent with gene conversion of the chromosomal alleles by the wild-type plasmid sequences. The loss of plasmid sequences from single prototrophs indicates that these transformants are the result

14 of marker rescue events, rather than survival of the damaged plasmid. This raises the possibility that the excess of Trp + transformants over His + and Ura ÷ transformants is due to marker rescue, rather than to enhanced repair. However, the majority of the Trp ÷ transformants carried YRpHUT, as indicated by their unstable His+Ura+Trp+ phenotype (Table 2), and by Southern hybridization of vector probes to plasmid-sized D N A (data not shown). Examination of the relative transformation and single Trp + prototroph data indicates that most of the "extra" Trp ÷ colonies do carry repaired plasmids. For instance, in the radI strain, at a psoralen modification level of 50 adducts per plasmid molecule, there was a 9-fold increase in the number of transformants measured in tryptophan omission medium, compared to transformants measured in uracil or histidine omission medium (Fig. 2). Thus, if the excess in Trp ÷ transformation were generated by marker rescue rather than plasmid repair, some 90% of the transformants should be single Trp ÷ prototrophs, rather than the 1.6% actually measured (Table 2). The larger number of transformants obtained in tryptophan omission medium is therefore due to more efficient plasmid D N A repair under these conditions. Discussion

The lesions induced by psoralen photoreaction of plasmid D N A include both monoadducts and interstrand crosslinks. While excision repair alone may be sufficient to repair monoadducts, no undamaged template strand is available at the crosslink sites, necessitating the involvement of other pathways for complete repair. Recombination-deficient rad52-I yeast cells were found to be more sensitive to psoralen crosslinks in their genomic D N A than RAD cells, while radl cells had intermediate sensitivity (Cassier et al. 1985). We have found that, as for chromosomal DNA, plasmid DNA repair depends upon RAD52 as well as upon the RAD1 and RAD3 excision repair genes. The most sensitive strain was the rad52 mutant, which had 37% relative transformation, corresponding to one lethal hit, at 3.5 psoralen adducts per molecule of Y R p H U T (Fig. 2). This compares with 30 adducts per lethal hit in the wild-type strain. Intermediate values were obtained for excision repair-deficient strains. The recombinational repair pathway thus plays a major role in the repair of psoralen damage in plasmid molecules. In recent studies of the URA3-containing plasmid YCp50, Magafia-Schwencke etal. (1991) reported that radl and rad52, but not rad6, cells were deficient in the repair of 8-methoxypsoralen damage. A similar dependence on RAD52, and other genes in this group, has been reported for the repair of double strand breaks (Perera et al. 1988; Glaser et al. 1990) or gamma irradiation (White and Sedgwick 1985)in plasmid DNA.

Differential repair of plasmid genes An unexpected finding was that the relative transformation rate with damaged plasmid depended upon the gene selected for. The levels of Trp ÷ transformants were

greater than those of Ura + or His + transformants. The influence of transformant selection conditions on the observed repair efficiency could reflect the initial distribution of psoralen adducts within the plasmid molecules. A lower level of psoralen adducts within the TRP1 gene than within HIS3 or URA3, for instance, could allow swifter repair in this segment and therefore resumption of TRP1 transcriptional activity. Cells placed under conditions of tryptophan starvation might therefore recover more efficiently than those grown in other selective media. We have not mapped the distribution of psoralen adducts within the photoreacted plasmids. However, inspection of the plasmid sequence does not suggest that there are large differences in the availability of psoralen reaction sites. The frequencies of the preferred TpA sites are 0.055, 0.067 and 0.069 sites per basepair in the HIS3, URA3, and TRPI genes, respectively; the frequencies of TA runs, which are potential hotspots for psoralen adduct formation (Sage and Moustacchi 1987) are 0.0062, 0.0077 and 0.0055 sites per basepair in these genes. The repair difference depended on RAD52, suggesting that recombination processes were involved. The repair difference was particularly large in the radl strain. A possible explanation for the enhanced differential is that, in the absence of RADI function, psoralen adducts are not excised, and are repaired by a recombination pathway that acts more efficiently under conditions of selection for TRPI than for the other plasmid genes. However, the rad3-2 strain, which is also excision repairdeficient, showed no greater a differential than RAD cells. The variation between the two excision repair-deficient strains may be due to different functions of the gene products in the repair process; Miller et al. (1984) have reported that yeast with this allele of RAD3 are capable of excising psoralen monoadducts, but not crosslinks. A factor which has been shown to regulate both excision repair efficiency and mitotic recombination frequency is the transcriptional activity of specific genes. Transcriptional activation is correlated with an increased excision rate of pyrimidine dimers in yeast (Terleth et al. 1989) and of several forms of DNA damage (Bohr et al. 1985; Bohr and Wassermann 1988), including psoralen adducts (Vos and Hanawalt 1987), in mammalian cells. Spontaneous recombination is also increased in response to R N A polymerase I (Keil and Roeder 1984) and R N A polymerase II (Thomas and Rothstein 1989) activity in yeast. The frequency of transformation to prototrophy is assayed under conditions of starvation for the selected nutrient, a situation in which the transcriptional activity of inducible genes is stimulated. Starvation for leucine has been reported to induce the spontaneous intrachromosomal recombination of repeated alleles of the inducible leu2 gene (Mink et al. 1990). In our experiments, differences in the transcriptional regulation of the HIS3, URA3, and TRPI genes could affect the repair of the plasmid by both excision and recombination pathways. While both HIS3 and URA3 are inducible, TRP1 is expressed constitutively (Miozzari et al. 1978); starvation

15 for histidine or uracil may produce different responses in transcriptional and repair activity than does starvation for tryptophan. Plasmid recombination

Psoralen damage was found to induce changes in the transformation phenotype, to auxotrophy for one or more nutrients, and to stability of the plasmid genes. These changes can be produced by recombination events such as gene conversion between plasmid and chromosomal alleles, or by plasmid integration. Psoralen damage induced single auxotrophy for each of the selected plasmid traits, but most frequently for histidine. Auxotrophy for one of the plasmid traits can be generated by conversion of the wild-type plasmid gene by the mutant chromosomal allele. This direction of gene conversion, in which the undamaged gene is the donor of genetic information and the damaged molecule is the acceptor, is expected to be the predominant event in D N A repair. Chemical or radiation damage in plasmids has been found to stimulate chromosome to plasmid conversion in bacteria; these events depend on recA function and the presence of homologous chromosomal sequences (Luisi-DeLuca et al. 1984; Chattoraj et al. 1984; Mudgett et al. 1990). Previous studies in yeast have shown that double strand breaks in plasmid and chromosomal DNA, produced by restriction endonuclease (Orr-Weaver and Szostak 1983) or H O endonuclease digestion (Nickoloff et al. 1989; Ray et al. 1988), act as acceptors in conversion events. The gene conversion is accompanied by reciprocal exchange in a fraction of the events ranging from 10% to 90%, depending upon the system examined. The plasmid conversions in this study were associated with crossing over in from 50 to 80% of the events in the R A D and rad3 strains (Table 3). The radl strain differed in that both the frequency of single auxotrophs and the fraction of reciprocal exchanges were lower. R A D 1 has recently been shown to function in certain forms of recombination, as well as in excision (Schiestl and Prakash 1988; Klein 1988). In a study of spontaneous intrachromosomal recombination of repeated genes, Aguilera and Klein (1989) found that gene conversion associated with crossing over depends on RAD1. We have previously found that the psoralen damage-induced integration of non-replicating plasmids into yeast chromosomes is reduced in radl cells (Saffran et al. 1992). The present study indicates a similar role for R A D 1 in the intermolecular recombination of replicating plasmids with chromosomes. Gene conversion in the opposite direction, producing singly prototrophic colonies, was also induced by psoralen photoreaction. Plasmid to chromosome gene conversion, in which the damaged D N A acts as genetic donor, was induced at levels comparable to, or higher than, chromosome to plasmid conversion. In this respect yeast cells differ from E. coli, in which damage-induced plasmid to chromosome conversion was infrequent (Mudgett and Taylor 1986). Single prototrophs differed from single auxotrophs

in that all were stable, and the majority of these transformants lacked either plasmids or integrated plasmid sequences. As plasmid was lost these should be classified as marker rescue, rather than D N A repair events. A possible mechanism for the production of single prototrophs is that psoralen damage induces plasmid fragmentation similar to that seen in chromosomal D N A ; the fragments produced replace homologous chromosomal sequences, as has been seen with restriction fragments (Rothstein 1983) and oligonucleotides (Moerschell et al. 1988) introduced into yeast cells. The pattern of damage-induced recombination varied among the plasmid genes we examined. Most of the recombination events involving TRP1 were plasmid to chromosome conversions producing single tryptophan prototrophs, In contrast, gene conversion of the H I S 3 loci on the plasmid and chromosome was induced in both directions, producing histidine auxotrophs as well as prototrophs. Both forms of recombination at the URA3 locus were relatively infrequent. Differing patterns of spontaneous plasmid-chromosome recombination involving these genes were also observed by Simon and Moore (1990), who reported that TRP1 plasmids transformed yeast cells by gene replacement, while transformation by URA3 plasmids occurred by integration. Thus, damage-induced recombination, like spontaneous recombination, appears to be locus- or allele-specific. Acknowledgements. We thank Drs. Rodney Rothstein and Corinne

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