Currt

Current Genetics (1983) 7:191-194

© Springer-Verlag 1983

Cloning of Photoreactivation Repair Gene and Excision Repair Gene of the Yeast Saccharomyces cerevisiae Akira Yasui I and Marie-Renee Chevallier 2 1 Medical Biological Laboratory, TNO, Lange Kleiweg 139, The Netherlands 2 Laboratoire de Genetique Physiologique, Institut de Biologic, Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, 67084 Strasbourg Cedex, France

Summary. The photoreactivation repair gene (PHR1) of the yeast Saccharornyces cerevisiae was cloned in a hybrid plasmid (pJDB207), which is able to replicate as a multicopy episome in S. cerevisiae and Escherichia coli cells. The size o f the DNA fragment found to have the photoreactivation activity was 3.0 kb, determined b y recloning o f the isolated fragment. In wild type cells transformed by the plasmid containing the PHR1 gene, the number o f DNA photolyase molecules was 15 times greater than in wild type cells with pJDB207 only. Using the same receptor strain the excision repair gene RAD1 was also isolated. The size o f the insert o f the DNA which complements excision repair deficiency in recipient yeast cells was 5.7 kb. The recipient cells after transformation with the plasmid containing RAD1 showed the same UV-sensitivity as wild type cells with pJDB207 only. Key words: Cloning of DNA repair genes - Photoreactivation repair - Excision repair

Introduction

1969). The defective gene is p h r l . The product of the gene RAD1 is necessary for the initial incision step of excision repair of pyrimidine dimers (Reynolds et al. 1981). In this paper we report the cloning o f the PHR1 as well as the RAD1 gene and the effects o f the genes in plasmids on yeast cell survival after UV-irradiation.

Materials and Methods Strains and Media. The S. cerevisiae strain Yal-27a (genotype: a

radl-1 phrl-1 ade2-1 his3 leu2) was constructed from the strain GRF18 (a, his3 leu2), agift from G. R. Fink, and from the strain Y2030XIIb (a, radl-1 phrl-1 ade2-1). The latter strain was constructed from PR74-35A (c~, phrl-1 met ade tyr leu his), a gift from M. Resnick, and X12-6B (a, radl-1 ade2-1) obtained from the Yeast Genetic Stock Center, Berkeley. The Escherichia coli strain used for transformation and amplification of plasmid DNA was BJ5183 ( F - , recBC sbcB endoI gal met str thi bio hsdR), a gift from B. Jarry. The cloning vector used was the plasmid pJDB207 (Beggs 1978). Yeast nitrogen base without amino acids (6.7 g/l; Difco) supplemented with 2% glucose was used as minimum medium. Complete medium consisted of 1% yeast extract, 1% peptone and 2% glucose. The liquid media were solidified with 2% agar (Difco).

Several different mechanisms for DNA repair have been reported in various organisms (Hanawalt et al. 1979). Photoreactivation repair and excision repair are effective repair mechanisms of UV-induced pyrimidine dimers. In photoreactivation process dimers can be monomerized by visible light after complex formation between dimers and-photolyase molecules (EC4.1.99.3) (Rupert 1975). In Saccharomyces cerevisiae a mutant completely deficient in photoreactivation has been isolated (Resnick

DNA Preparation and Digestion. Plasmid DNA from E. coli was

Offprint requests to." A. Yasui Abbreviation: UV, Ultra-violet light of 254 nm wavelength

Transformation. Yeast was transformed by the method of Hin-

extracted by the method of Clewell (1972). Themethod for rapid preparation of yeast DNA (Struhl et al. 1979) was used. In recloning procedures DNA was electrophoresed on horizontal agarose gels as previously described (Chevallier et al. 1980). The DNA fragment of interest was then cut out, electro-eluted, and precipitated with tRNA as a carrier. The DNA pellet was resuspended in ligation buffer, incubated with T4DNA ligase for 10 h at 20 °C, and used to transform E. coli recipient cells. Restriction endonuelease digestions and ligation treatment of DNA were carried out as recommended by the manufacturers (Bethesda Research Laboratories and Boehringer Mannheim).

nen et al. (1978). Zymolyase 60,000 (Kirin Brewery Ltd., Japan)

192

A. Yasui and M.-R. Chevallier: Cloning of Photoreactivation and Excision Repair Gene

pBR322

Irradiation Conditions. All experiments were performed under

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red light to prevent uncontrolled photoreactivation. The cell concentration during UV-irradiation and photoreactivation was 1 x 106 cells/ml buffer. Other experimental conditions were the same as those previously reported (Yasui and Laskowski 1975). The results depicted in the figures are average values obtained in three to five independent identical experiments.

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was used at a final concentration of 50 #g/ml to prepare the spheroplasts. E. eoli was transformed by the method of Cohen et al. (1972).

Growth Conditions and Cell Preparation for the Determination of Photoreaetivation Efficiency and UV-Sensitivity. The plasmid infected cells were transfered to a minimum medium plate supplemented with adenine and histidine but without leucine and incubated for 72 h at 30 °C. The ceils were then suspended in buffer (0.05 M KH2PO4: PH4.8) and washed twice in the same buffer. In order to obtain single cells, the suspension was then sonicated for ten seconds. This sonication procedure had no effect on the survival and UV-sensitivity of the cells.

UV-Irradiation. The UV source was a low pressure mercury lamp (Osram HNS12, FRG) with a maximum emission at 254 nm wavelength. The dose rate was 0.42 j m - 2 s -1 determined by an Opto-meter 80 x (United Detector Techn., Santa Monica, CA, USA), which had been calibrated for the wavelength. Photoreactivation. For the isolation of photoreactivable cells a continuous illumination from an Osram L-80-70 lamp (FRG) was used. The distance between the cell suspension in a small glass dish and the lamp was 10 cm. In control experiments it was shown that 30 min illumination under these conditions gives the maximum level of survival increase by photoreactivation to the cells of radl PHR genotype, which had been irradiated with the dose of 16.8 j m - 2 s -1. To determine the number of photolyase molecules per cell an intense light flash was used (Yasui and Laskowski 1975). An electronic flash unit (Rollei E36RE, FRG) delivers a light flash of 1/800 s duration. The flash of light was given to the cell suspensions from a distance of 5 cm. It is known that all complexes between photoreaetivable dimers and photolyase molecules present at the moment of the light flash can be split by one flash of light (Yasui 1976).

Isolation o f DNA Fragments Able to Restore Photoreactivab ility or UV-R esistance in Strain Ya l-2 7a A yeast gene bank was made by Losson and Lacroute using the high copy number hybrid bacterial yeast plasmid pJDB207 as vector. The construction of the plasmid pool has been described by Losson and Lacroute (1981). After transformation of the yeast strain Yal-27a by the gene bank, Leu + transformants were selected on appropriately supplemented minimal medium plates without leucine. The use of the double m u t a n t was especially convenient since in the radl background we can obtain large photoreactivation by a short time of illumination, and UV-resistance is tested by irradiation with a large UV dose. From each of the 1 5 0 - 2 0 0 colonies of transformed cells a cell suspension was made, which was then sonicated to obtain single cells. The suspensions were irradiated with UV-doses of 16.8 Jm - 2 and 50 Jm - 2 , respectively and plated on complete medium plates. An aliquot of each suspension irradiated with 16.8 Jm - 2 was illuminated 30 min for maximal photoreactivation and then plated. One out of ten suspensions gave more colonies on plates after photoreactivation than without the treatment. With similar frequency we found survivors from cells irradiated with 50 Jm - 2 , whereas most cell suspensions showed no survivors after this irradiation. Yeast DNA was isolated from these selected clones and used to transform E. coli cells. Plasmid DNA from the E. coli cells was analysed by restriction digests. The plasmid A8-3 complemented the photoreactivation deficiency of yeast strain Yal-27a. A8-3 has an insert of 4.0 kb. Another plasmid, called A2-2, conferred UV-resistance to the yeast strain Yal-27a. The A2-2 insert is 5.7 kb long. All L e u - cells derived from A8-3 or A2-2 transformants showed neither photoreactivation nor UVresistance.

Restriction Mapping o f the Plasmids A8-3 and A2-2 The restriction map of the insert of A8-3 is given in Fig. 1. The following enzymes did not cut the insert; BamHI, EcoRV, HindIII, HpaI, SalI, SphI. Ttae restriction map of the insert of A2-2 is depicted in Fig. 2. No site was found for SalI, PstI, SphI, BamHI and AvaI.

A. Yasui and M.-R. Chevallier: Cloning of Photoreactivation and Excision Repair Gene UV d o s e , 0

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Fig. 3. Survival after UV-irradiation (closed symbols) and UVirradiation plus photoreactivation (open symbols). For photoreactivation 5 flashes were given to the strain GRF18[pJDB207] (Azx) and only one flash was given to the strain GRF18[AS-3] (o e) after UV-irradiation

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Figure 3 shows survival curves after UV-irradiation and photoreactivation. Wild type cells of GRF18 were transformed by pJDB207 or by the isolated plasmid A8-3. As seen in this figure the effect of photoreactivation by one flash of light to the cells carrying plasmid A8-3 is at least 3 times larger than that of five flashes to the cells carrying plasmid pJDB207 if the dose decrements (the fraction of UV-dose whose effect has been annulled by photoreactivation) are compared. As previously described (Harm et al. 1971; Yasui 1976) the dose decrement by one photoreactivation flash is proportional to the number of photolyase molecules in a cell, if there is an excess of substrate for the enzymes. Five flashes were given, with sufficient time intervals, to enable enzymes to form new complexes with dimers. Our results show that the plasmid A8-3 in a yeast cell increases the concentration of photolyase molecules at least 15 times. Since the concentration of photolyase molecules was determined to be 350 in a haploid yeast cell without the plasmid A8-3 (Yasui 1976), there are at least 5.3 x 103 enzyme molecules in a yeast cell habouring the plasmid A8-3.

Dose Effect Curve of the Cells Carrying the Plasmid A2-2 Figure 4 depicts the survival curves of yeast strains YA127a[pJDB207], Yal-27a[A2-2] and GRF18[pJDB207]. As seen in this figure, presence of the plasmid A2-2 in strain Yal-27a confers the same sensitivity as have wild type cells carrying plasmid pJDB207.

Recloning of the Plasmid A8-3 Photoreactivability of yeast Ya1-27a cells which contained different fragments of the A8-3 insert were tested. The photoreactivation repair gene was found situated between the two PvulI sites. This DNA fragment is 3.0 kb long.

Discussion -3

10

10

Fig. 4. Survival after UV-irradiation of the strains Yal-27a[pJDB207] (e), Yal-27a[A2-2] (m) and GRF18[pJDB207] (A)

By comparing the phtotoreactivation efficiencies of transformed cells we determined that the presence of the plasmid A8-3 increased the concentration of photolyase molecules by at least a factor of 15 over the level normally found in yeast cells. This increased concentration of photolyase molecules due to the plasmid-borne photoreactivation gene agrees with that found in E. coli cells, into which the bacterial photoreactivation gene had been introduced (Rupert and Sancar 1978). In spite of the

194

A. Yasui and M.-R. Chevallier: Cloning of Photoreactivation and Excision Repair Gene

high concentration of photolyase molecules which form complexes with pyrimidine dimers in UV-irradiated cells we found no effect o f the enzymes on survival o f the cells, if cells were incubated in the dark after UV-irradiation (Fig. 3). This m a y indicate that photolyase molecules do not interfere with DNA repair o f other repair mechanisms. W - s e n s i t i v i t y of the strain Yal-27a[A2-2] is the same as that of wild type cells carrying plasmid pJDB207. This may indicate that there is no increased production of the RAD1 gene product over that found in the wild type or that an increased production o f the RAD1 protein does not enhance the UV-resistance compared to wild type level. However, it fully complements the defect o f m u t a n t allele r a d l on chromosomal DNA. We introduced A8-3 or A2-2 plasmids into E. coli cells with photoreactivation repair (phrA) or excision repair (uvrA and uvrB) deficiencies. Although the cells had a high copy number o f the plasmid A8-3, we could not detect photoreactivabllity o f the cells. Nor did plasmid A2-2 change the UV-sensitivity o f either excision repair defective strain of E. coli. Since it is known that yeast photolyase molecules, which are different from photolyase molecules in E. coli cells, can repair pyrimidine dimers in bacterial DNA in vitro, lack of photoreactivation in E. coli cells b y the A8-3 plasmid may indicate a lack o f expression of the yeast gene in these cells. It remains to be determined whether the product o f RAD1 gene complements the uvrA or uvrB gene products in vitro, which are also responsible for the incision step in excision in E. coli. Cloned repair genes should help to understand repair mechanisms and the regulation o f the synthesis o f repair proteins. Furthermore, they will provide the means to obtain large quantities o f repair proteins.

W. Laskowski for critical reading of the manuscript and to Mme B. Werl6 for the expert technical assistance.

Note Added

During the l l t h international conference on yeast genetics and molecular biology held in Montpellier in Sept. 1982, in which the results reported in this paper were presented, we heard that J. Johnson, F. Hilger, D. Schild and R. Mortimer have also cloned PHR1 gene of Saceharomyees cerevisiae. The cloning of the RAD1 gene was also reported in the conference by the Stanford group (L. Naumovski, E. Yang, G. Pure and E. C. Friedberg) and by J. F. Lemontt.

References

Beggs JD (1978) Nature (London) 275:104-109 Chevallier MR, Bloch JC, Lacroute F (1980) Gene 11:11-19 Clewell DB (1972) J Bacteriol 110:667-676 Cohen SN, Chiang ACY, Hsu L (1972) Proc Natl Acad Sci USA 69:2110-2114 Hanawalt PC, Cooper PK, Ganesen AK, Smith CA (1979) Annu Rev Biochem 48:783-836 Harm W, Rupert CS, Harm H (1971) In: Giese AC (ed) Photophysiology. Academic Press, New York London, pp 279-324 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929-1933 Losson R, Lacroute F (1981) Mol Gen Genet 184:394-399 Resnick MA (1969) Photochem Photobiol 9:307-312 Reynolds RJ, Love JD, Friedberg EC (1981) J Bacteriol 147: 705 -708 Rupert CS (1975) In: Hanawalt PC, Setlow RB (ed) Molecular Mechanisms for Repair of DNA. Plenum Press, New York, pp 73-87 Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035-1039 Yasui A, LaskowskiW (1975) Int J Radial Biol 28:511-518 Yasui A (1976) PhD Thesis, Freie Universit~it Berlin

Communicated by C. P. Hollenberg

Acknowledgements. We should like to thank Prof. F. Lacroute for providing us with the gene bank. We are also grateful to Prof.

Received January 24 / February 28, 1983

Cloning of photoreactivation repair gene and excision repair gene of the yeast Saccharomyces cerevisiae.

The photoreactivation repair gene (PHR1) of the yeast Saccharomyces cerevisiae was cloned in a hybrid plasmid (pJDB207), which is able to replicate as...
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