Curt Genet (1987)11:321-326

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Induced cellular resistance to ultraviolet light in Saccharomyces cerevisiae is not accompanied by increased repair of plasmid DNA C. I. White I and S. G. Sedgwick

Genetics Division, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK

Summary. Many reports show that resistance of Saccharomyces cerevisiae to a large UV dose can be enhanced by pre-induction with a smaller one given some hours before. This work tests if such increased cell survival is associated with increased DNA repair on UV damaged plasmid transformed into yeast. There was no change in transformation efficiency of UV-damaged plasmid DNA under conditions where RAD cell survival increased 5fold, and where radl-1 and rad6-1 survival increased 2-fold. It is concluded that DNA repair activity involving the RAD6 and RAD3 pathways is either not inducible or is unable to work on plasmid DNA. It is suggested that the enhancement of cellular survival detected may be based on changes in cell-cycle behaviour which permit cells generally proficient in repair a greater chance to recover. Key words: Saccharomyces cerevisiae - Inducible repair - Plasmid transformation

Introduction

The first indication that DNA repair capacity could be increased in response to the amount of DNA damage was provided in 1953 by Weigle. He showed that the survival and induced mutagenesis of UV-irradiated lambda phage was increased by infecting E. coli which itself had been irradiated. Since then a common feature of many investigations into inducible repair has been to induce a cell with one dose of DNA damage. The idea is to test

1 Present address: Rosentiel Basic Medical Sciences Research Center, Brandeis University,Waltham, MA 02256, USA Offprint requests to: S. G. Sedgwick

whether the cell is then more able to withstand a second challenge dose, or better able to reactivate damaged phage, viral or plasmid DNA. In the yeast, Saccharomyces cerevisiae, enhanced cell survival in such split-dose experiments provides evidence consistent with inducible recovery processes (Patrick and Haynes 1968; Ferguson and Cox 1980; Eckardt et al. 1978; reviewed by Siede and Eckardt 1984). The need for protein synthesis for maximal survival after a single dose is also consistent with this idea (Heude et al. 1975; Siede et al. 1983; Lee 1983). In yeast, DNA repair processes have a major influence on cellular resistance to radiation and so are candidates for being the inducible processes enhancing cell survival. Yeast radiation repair activity can be subdivided into three pathways, two of which repair most UV damage (reviewed by Game 1983). One is the RAD3 pathway encompassing nucleotide excision repair. The other is the RAD6 pathway, and although its action is not clear, causes radiation4nduced mutagenesis, rev3 is another member of the RAD6 group, it is deficient in induced mutagenesis, but is relatively insensitive to UV killing (Lemontt 1971). Thus rev3-1 presumably has a more specific defect in the mutagenic response than is caused by the pleiotrophic rad6-1 mutation (reviewed by Lawrence 1982). The evidence that UV damage stimulates increased RAD3 and RAD6 types of repair is fragmentary. In splitdose experiments Ferguson and Cox (1980) identified a UV4nducible activity enabling a greater fraction of pyrimidine dimers to be excised after a challenge dose of UV. In rev2 ts mutants, which belong to the rad6 group, normal levels of survival and mutagenesis were reduced by post-irradiation incubation at non-permissive temperature (Siede et al. 1983). At the permissive temperature protein synthesis was needed for full resistance and mutagenesis to be developed. However in split-dose experiments protein synthesis was still required for maxi-

322

c.I. White and S. G. Sedgwick: Enhanced UV resistance of Saccharomyces cerevisiae

real resistance and mutagenesis after the second challenge dose. This result raises the possibility that it is ongoing, rather than de novo protein synthesis which is required for maximal UV survival. At the molecular level there is damage-induced expression of a number of DNA repair related genes. The R A D 5 4 gene transcript is induced 3 - 5 fold after X-irradiation of yeast cells (Emery et al. 1984), and Peterson et al. (1985) have shown UV inducible transcription of the DNA ligase gene, CDC9. Barker and Johnston (1983) have observed this CDC9 response to both UV and methylmethane suphonate using CDC9-lacZ fusions. A 3-fold induction of RAD2-lacZ expression was also found after UV irradiation (Robinson et al. 1986). In addition several Lrv inducible sequences have been identified by their inducible transcripts or their expression in /ac-fusion vectors (McClanahan and McEntee 1984; Ruby and Szostak 1985). However the function of these genes remains u n k n o w n although some transcripts have been shown to originate from the yeast Ty element (Rolfe et al. 1985). The experiments described here first demonstrate the enhancement of cellular radiation resistance and the effects of radl-1, rev3-1 and red6-1 mutations. They then assay whether these cellular changes are accompanied by enhanced DNA repair activity. The approach used was to compare the transformation efficiencies of UV irradiated plasmid DNA in cells whose resistance had been enhanced or not. Previous work had shown that transformation efficiency of irradiated DNA was determined by the same R A D 3 and R A D 6 repair systems which also operate on genomic DNA (White and Sedgwick 1985). Thus transformability of UV irradiated plasmid is equated here with plasmid repair and survival.

Materials and methods

Materials. General media constituents were from Difco Laboratories. Other chemicals, antibiotics and amino acids were from Sigma Chemical Company or BDH Chemicals, and were of analytical g~ade. Media. Complete medium based on yeast extract-peptone glucose (YEPD) and synthetic minimal medium and supplements were as described in Sherman et al. (1971). Yeast. Listed in Table 1 are the haploid S. cerevisiae mutants used in this work. Strains C4-5 and C6-3 are progeny of matings between YNN27 (from Dr. G. Banks) and strains kindly sent by Dr. J. Game. C9-4 and C11-1 were isolated as spontaneous lys2 mutants from strains YNN27 and C6-3 respectively, using aaminoadipate selection (Chatto et al. 1979). These cells were shown to be lys2 by crossing to appropriate tester strains. The rev3-1 stock was a gift from Dr. B. S. Cox. Piasmid DNA. The yeast shuttle vector YRpl2 has approximately 6,900 bp and consists of pBR322 with the yeastARS1-TRP1

and URA3 genes inserted into the EcoRI and Aval sites respectively (Schcrer and Davis 1979). Plasmid pC3-1 consists of the yeast centromeric vector, YCp50 (from Dr. C. Mann), with a 4,800 bp insertion containing the yeast LYS2 gene. This plasmid has 12,840 bp and contains CEN4, the centromeric DNA of yeast chromosome 4, ARS1, URA3 and LYS2 genes. The 4,800 bp L YS2 fragment originated as a Sall fragment from plasmid pDP13 (Eibel and Phillipsen 1983) inserted into the SalI site of YCp50. Plasmid DNA was prepared from transformed recA56 E. coli by the alkaline lysis procedure and purified by equilibrium density gradient centrifugation in CsCl-ethidium bromide (see Maniatis et al. 1982). DNA was stored at - 2 0 ° C in 10 mM Tris-HC1 (pH 7.4), 2.5 mM EDTA at approximately 250 ~zg/ml.

UV irradiation. Yeast cells: mid-log phase cells at approximately 107 cells/ml were harvested by membrane filtration and resuspended in sterile distilled water at 106 ceUs/ml.This suspension was UV irradiated in plastic tissue culture dishes with constant agitation. The UV source was a Hanovia low pressure germicidal lamp calibrated before each experiment with a UVX-Radiometer (Ultraviolet Products Ltd., Cambridge, UK) Appropriate dilutions were plated on YEPD agar and cell survival determined from colony counts after 3-5 days growth at 32 °. All manipulations were carried out in semi-darkness to minimise photoreactivation. DNA: plasmid DNA was UV irradiated at a concentration of 250 ug/ml in 10 mM Tris-HCl (pH 7.4), 2.5 mM EDTA. 15-30/~1 droplets of DNA were exposed on the surface of plastic Petri dishes to UV from a Hanovia low pressure germicidal lamp giving an incident flux of 3.3 J/m2/s. The lamp was calibrated before each use. Split-dose irradiatons: midqog phase ceils were UV-irradlated in sterile water and resuspended in prewarmed YEPD medium for incubation at 32 °C. The cells were then harvested and resuspended in sterile water at a density of 106/ml. Aliquots were removed and plated to determine the concentration of viable cells. The remainder were exposed to the second UV dose and plated, or incubated for 7 h in YEPD containing 5 #g/ml cycloheximide at 32 °C, washed and then plated for survival. Survival was calculated from the concentration of viable cells after the second dose, relative to that immediately before. These experiments were carried out in two ways: (1) The time of incubation between doses was varied with constant initial dose. This enabled the time course for the development of UV resistance to be followed. (2) A constant time between doses was used and the initial dose was varied to determine the dose-response for increasing resistance. Transformation. Yeast cells were transformed by the LiC1 method (Ito et al. 1983). Each transformation consisted of approximately 108 cells, 3-4 tzg plasmid DNA, 20 #g denatured salmon sperm DNA, and was plated directly on minimal medium plates with the neccessary supplements. The concentration of plasmid DNA was determined to be non-saturating in terms of transformants per/~g DNA for each of the strains used. Colonies transformed to URA+ or LYS+ were scored after 4 to 5 days growth at 32°. Control transformations with no plasmid DNA showed no observable spontaneous reversion of selected phenotype.

Results Conditions for enhancing cellular resistance to a challenge UV dose by pre-irradiation with a smaller inducing dose

C. I. White and S. G. Sedgwick: Enhanced UV resistance of Saccharomyces cerevidae

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1. The effect of varying the inducing dose given to RAD (o) rev3-1 (V) ceils 3 h before receiving a challenge dose of J/m 2. The figure shows the survival of the ceils to the secdose

Fig. 2. The effect of varying the inducing dose given to rad6-1 (£x) and tad1-1 (o) cells 3 hrs before receiving a challenge dose of UV radiation. Challenge doses were 22.5 J/m 2 for rad6-1 and 5.5 J/m 2 for radl-1. The figure shows the survival of the cells to the second dose

were first established. One form of split-dose experiment assayed the inducing effects of a range ofintial UV doses. Three hours incubation in YEPD medium followed before exposure to a second challenge UV dose of 220 J/m 2 . As expected from numerous earlier experiments the resistance of RAD ceils to this challenge dose of UV was increased by irradiation with the lower UV dose given 3 h before (Fig. 1). An approximate 5-fold increase in UV resistance was produced by pre-irradiation with 15-35 J/m 2 UV. rev3-1 cells showed a 10-fold increase in radioresistance when given 5 J/m 2 3 h previously (Fig. 1). Similar experiments (data not shown) had shown that maximal increases in cell survival had developed within 2.5 to 3 h of incubation between irradiations. The same experimental regime caused a smaller 2-fold increase in the survival of rad6-1 and tad1-1 mutants to a subsequent challenge dose (Fig. 2). Because of the greater UV sensitivity of these mutants smaller initial and challenge doses were employed. In rad6-1 cells maximal resistance to a challenge dose of 22.5 J/m 9- was produced with 2 - 3 J/m 2 pre-irradiation; in the radl-1 mutant approximately 2-fold resistance to a challenge dose of 5.5 J/m 2

was produced within a very narrow range of conditioning doses around 0.5 J/m 2 UV. UV irradiated plasmid DNA was next used to test if the increased cellular survival also enhanced plasmid transformability, and therefore repair of plasmid DNA. Thus cellular resistance to UV was increased in a split dose experiment. The cells were given an initial UV dose and incubated 2.5 h. The doses used were 27 J/m 2 for the RAD cells, 4 J/m 2 for rad6.1, and 0.55 J/m 2 for radl-1 mutants. Control cells were not irradiated but otherwise were manipulated identically. After the postirradiation incubation one portion of each culture received a challenge dose of UV. Assays of colony forming ability verified the expected increase in resistance of the pre-irradiated cells to a challenge dose of UV (data not shown but see Table 2). Another portion of each culture was made competant for transformation. Competant ceils were transformed with plasmid DNA which had been given a range of UV doses. The transformations efficiencies of this irradiated DNA were unchanged by pre-irradiation of the cells (Fig. 3). Thus the increased cell survival in RAD, tad1-1 and rad6-1 cells

C.I. White and S. G. Sedgwick: Enhanced UV resistance of Saccharomyces cerevisiae

324 Table 1. Haploid yeast strains Strain

Genotypes

YNN27 C4 5

RAD, trp1-289, ura3-52 rad1-1, trp1-289, ura3-52 rad6-1, trp1-289, ura3-52, leu2 RAD, trp1-289 , ura3-52, lys2 rad6-1, trp1-289, ura3-52, lys2 rev3-1, hisS-2, arg4-17 , lysl-11

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Table 2. The effect of plasmid transformation on the resistance of RAD yeast to 180 J / m 2 UV Cells

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had no effect on DNA repair as judged by transformation efficiencies in induced and non-induced cells by UV-damaged plasmid. It was possible that the transformation protocol was itself sufficient to enhance the DNA repair capacity of the cells. If this were so, all cells would have been induced and no differences in plasmid survival would be expected. This possibility was tested by seeing if the transformation procedure increased the UV resistance of R A D cells in a split-dose irradiation experiment. Control cells, which were not made competent, received 0 or 25 J/m 2 UV, were incubated 2.5 h, given a challenge dose of 180 J/m 2, and plated on YEPD agar. As expected, the pre-irradiation treatment caused and increase in resistance to the second dose, in this case 15-fold (Table 2, lines 1 and 4). Portions of the same culture irradiated with 0 or 25 J/m 2 UV and incubated 2.5 hours, were made competent and transformed with either unirradiated or irradiated plasmid DNA. The resistance of these cells to a UV challenge dose of 180 J/m 2 was then determined by colony forming ability on YEPD agar. Yeast which had not received any pre4rradiation showed 5 to 6 fold increases in cellular resistance after passing through the transformation proceedure with normal and damaged plasmid DNA (Table 2, lines 2 and 3). The same proceedure was applied to yeast which had been pre-irradiated with 25 J/m 2 UV. The 15 fold resistance which these cells developed was reduced to an 11.7 fold increase after undergoing the transformation proceedure with undamaged plasmid DNA (Table 2, line 5). When UV irradiated plasmid was used there was little reduction in resistance (Table 2, line 6). Thus the transformation process itself can cause some changes in cellular resistance but these are smaller than those elicted by dose fractionation.

Discussion The aim of these experiments was to look for inducible DNA repair activity in yeast. From our knowledge of inducible bacterial repair systems sub-lethal UV doses might be expected to trigger synthesis of DNA repair proteins (reviewed by Walker 1984; Sedgwick 1986). At the cellular level these repair proteins might confer extra cellular resistance to a second challenge dose. These experiments confirm several earlier demonstrations which indeed show that radiation resistance in yeast is increased by pretreatment with conditioning doses of UV. The problematic point of this and previous work is whether the UV resistance enhanced in this way is directly attributable to inducible DNA repair activity or to some other cellular recovery process. The plasmid transformation assay of DNA repair proficiency (White

C. I. White and S. G. Sedgwick: Enhanced UV resistance of Saccharomyces cerevisiae and Sedgwick 1985) used here avoids the inherent complications of split-dose systems because the stimulus for induction can be separated from the substrate of the DNA repair assay. The results obtained with this assay show that conditions which produced cellular resistance to UV in R A D yeast did not influence repair proficiency on UV-irradiated plasmid DNA. Plasmid survival was also unaffected in radl-1 and rad6-1 cells where similar experimental regimes produced smaller increases in cellular resistance. These experiments therefore indicate that DNA repair by excision and RAD6 pathways in yeast is not UV-inducible, at least for repair of incoming plasmid DNA. Of the earlier reports of increased cellular resistance produced by dose fractionation, only one (Ferguson and Cox 1980) actually made direct measurements of increased DNA repair activity. In that example, split-dose irradiation increased the fraction of pyrimidine dimers excised after the second challenge dose of UV radiation. Also the onset of this repair was more rapid. Accordingly these workers found no enhancement of cellular survival by dose fraction of excision defective rad4 and rad16 cells. However there are other indications that changes in excision repair are not only events responsible for the enhancement of cellular survival. Firstly, by using smaller conditioning doses it was possible here to detect some enhancement of survival by dose fractionation in radl-1 yeast which lack any excision repair. In making this comparision of R A D with tad1-1 mutants, very different dose ranges had to be used to compensate for the radiosensitivity of the repair-deficient mutants. Thus these different cells may be using different residual repair activities to cope with differing degrees and types of DNA damage. Therefore it may not be valid to compare the recovery events in wild-type cells with those in the mutant strains. Should similar mechanisms exist it must be supposed that some process stimulating cellular recovery is activated at a given level of persisting DNA damage. In repair-deficient cells this level would be reached at lower doses than in wild-type. Despite these provisos the result with radl-1 yeast shows that some form of enhanced cellular recovery can occur independently of excision repair. Results of experiments with mutants affected in the RAD6 repair pathway, which does not include nucleotide excision, also support this point. For instance little or no resistance was produced by split-dose irradiation of rad6-1 yeast or rev2 ts cells at high temperature (Siede et al. 1983). In rev3-1 mutants, which also belong to the rad6 epistatic group but are more radioresistant, enhanced cellular survival in split-dose irradiation experiments was found. One could theoretically conclude that some RAD6-dependent, REV3 independent repair, in addition to excision repair, was responsible for enhanced UV resistance. However it is notable that low or absent levels of enhanced UV

325

resistance appear to be a general feature the tad1-1, rad6-1 and rev2 ts radiosensitive mutants, whereas radioresistant RAD and rev3 cells displayed increased UV resistance in split-dose experiments. Thus the ability to enhance UV resistance may depend in a secondary way on the cell's overall repair capacity rather than on any one specific DNA repair system. For example, changes in cell-cycle behaviour might allow greater opportunity for repair of any type to operate (Chanetet al. 1973). Such changes may have more effect on the repairability of genomic DNA compared with small plasmid molecules. This might perhaps explain the discrepancy between the finding of more efficient excision repair of genomic DNA after split-dose irradiation (Ferguson and Cox 1980) compared to the lack increase in the repair of plasmid DNA found here. In conclusion, the inducible UV resistance detected in Saccharomyces cerevisiae may be due to a number of causes and is not directly correlated with increased ability to repair UV irradiated DNA. Even though inducible repair gene expression has been reported by others it is worth remembering that these activities must perform at rate limiting steps for their inducibility to be manifested at the cellular level. Acknowledgements. We are indebted to Drs. B. Cox, J. Game and C. Mann for yeast stocks and plasmids. Dr. E. Freidberg kindly communicated results before publication. We thank G. R. Banks and L. Johnston for valuable advice in the preparation of this manuscript and our reviewer for constructive advise on its revision.

References Barker DG, Johnston LH (1983) Eur J Biochem 134:315-319 Chanet B, Williamson DH, Moustacchi E (1973)Biochim Biophys Acta 324.290-299 Chatto BB, Sherman F, Azubalis DA, Fjellstadt TA, Mehnert D, Ogur M (1979) Genetics 93:51-65 Eckardt F, Moustacchi E, Haynes RH (1978) In: Hanawalt PC, Friedberg EC, Fox CF (eds) ICN-UCLA Symposium on Molecular and Cellular Biology 9:421-423 Eibel H, Philippsen P (1983) Mol Gen Genet 191:66-73 Emery HS, Schild D, Kellog DE, Mortimer RK (1984) Proceedings of the XII International Conference on Yeast Genetics and Molecular Biology, Edinburgh Ferguson LR, Cox BS (1980) Mutat Res 69:19-41 Game J (1983) Radiation-sensitive mutants and repair in yeast. In: Spencer JFT, Spencer DM. Smith ARW (eds) Yeast genetics. Fundamental and applied aspects. Springer, New York Berlin Heidelberg, pp 109-137 Heude M, Chanet R. Moustacehi E (1975) Mutat Res 28:37-45 lto H, Fukuda Y, Murata K, Kimura A (1983) J Bacteriol 153.163-168 Lawrence CW (1982) Adv Genet 21:173-254 Lee MG (1983) PhD Thesis, Council for National Academic Awards, London Lemontt JF (1971) Genetics 68:21-33

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C.I. White and S. G. Sedgwick: Enhanced UV resistance of Saccharomyces cerevisiae

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY McClanahan T, McEntee K (1984) Mol Cell Biol 4:2356-2363 Patrick MH Haynes RH (1968) J Bacteriol 95:1350-1354 Peterson TA, Prakash L, Osley MA, Reed SI (1985) Mol Cell Biol 5:226-235 Robinson G, Nicolet C, Kalainov D, Freidberg EC (1986) Proc Natl Acad Sci USA 83:1842-1846 Rolfe M, Spanos A, Banks GR (1985) Nature (London) 319: 339-340 Ruby SW, Szostak JW (1985) Mol Cell Biol 5:75-84 Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:49514955 Sedgwick SG (1986) Microbiol Sci 3.76-83

Sherman F, Fink GR, Lawrence CW (1971) Methods in yeast genetics. Laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Siede W, Eckardt F (1984) Mutat Res 129:3-11 Siede W, Eckardt F, Brendel M (1983) Mol Gen Genet 190:406412 Walker GC (1984) Microbiol Rev 48:60-93 Weigle JJ (1953) Proc Natl Acad Sci USA 39:628-636 White CI, Sedgwick SG (1985) Mol Gen Genet 201:99-106

Communicated by B. S. Cox Received June 4, 1986