211

Mutation Research, 263 (1991) 211-215 © 1991 Elsevier Science Publishers B.V. 0165-7992/91/$ 03.50 ADONIS 016579929100065F MUTLET 0512

Transformation of Saccharomyces cerevisiae with UV-irradiated singlestranded plasmid Zoran Zgaga Faculty of Food and Biotechnology, University of Zagreb, 41000 Zagreb (Yugoslavia) (Received 1 March 1991) (Revision received 19 March 199 I) (Accepted 26 March 199 l)

Keywords: Yeast; Single-stranded plasmids; UV irradiation; Rad genes; Mutation induction

Summary UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J / m E) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad + strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the radl8 or tad52 mutations influenced the efficiency o f transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and L E U 2 genes (psLU) gave rise to up to 5°70 L e u - transformants among selected Ura + ones in the repair-proficient strain. Induction of Leu - transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged singlestranded DNA in yeast.

Three pathways for dark repair of UV-induced damage to DNA have been described in yeast (Haynes and Kunz, 1981; Moustacchi, 1987; Friedberg, 1988). Excision repair is thought to eliminate most of the damages, reestablishing the original genetic formation. Some lesions can be repaired by recombination involving either sister chromatids or homologous chromosomes and Correspondence: Dr. Z. Zgaga, Faculty of Food and Biotechnology, University of Zagreb, Pierrottieva 6, 41000 Zagreb (Yugoslavia).

resulting in crossing-over and gene conversion. The third type of repair process is called mutagenic or error-prone repair and is characterized by the appearance of new sequence alterations. The study of DNA repair in yeast was greatly facilitated by the isolation of numerous repair mutants that were shown to fall into 3 epistasis groups with respect to survival after UV irradiation. The mutants from the R,4D3 group are deficient in excision repair, and the mutants in the R A D 5 2 epistasis group in recombinational repair. The R A D 6 epistasis group is phenotypically very heterogeneous and involves

212

the genes that are required for mutagenic repair, such as R A D 1 8 and R E V 3 . It was suggested that the initial UV-induced lesion leading to both mutagenic and recombinational repair consists of 2 closely spaced pyrimidine dimers residing in opposite DNA strands (James et al., 1978; Fabre, 1981). Elimination of one of the dimers by excision repair would often leave the remaining dimer in single-stranded DNA, in front of a gap. This lesion cannot be repaired by excision, but is repaired by either error-prone DNA synthesis (James et al., 1978) or recombination (Fabre, 1981). Reynolds (1987) has shown that closely opposed dimers can be induced in yeast chromosome following UV irradiation, and that such structures are subjected to cellular repair processes. Thus the repair of lesions in single-stranded DNA may be responsible for most, if not for all, genetic changes arising as a consequence of UV irradiation. The process of postreplicative repair was studied in yeast strains deficient in excision repair, and was shown to de-

pend on the function of the R A D 6 gene. Postreplicational repair is greatly inhibited also in radl8 and rad52 mutants, but not in rev3 strain (di Caprio and Cox, 1981; Prakash, 1981), which is otherwise entirely deficient in UV-induced mutagenesis (Lemontt, 1971). One way of studying the repair of specific DNA lesions in yeast is transformation of competent cells with plasmid DNA previously modified in vitro. Integrative plasmids were extensively used to study the mechanism of double-strand break repair in yeast (Orr-Weaver et al., 1981) and replicative plasmids for the repair of UV-induced lesions (White and Sedgwick, 1985; Keszenman-Pereira, 1990). The transformation of yeast with singlestranded plasmids (Singh et al., 1982) makes it possible to study the repair processes operating on extracellularly damaged single-stranded DNA. In this study yeast replicative plasmids were isolated from E. coli in single-stranded form, irradiated with UV light in vitro and used to transform a repair-proficient strain and several repair-deficient yeast strains. Materials and methods

Plasmids

Sal I

I~ .oo

o,j//

~ S s I |iXho I)

Plasmid pLS42 (J.M. Buller, Saclay) was constructed by integrating the A R S 1 CEN4 fragment (1.37 kb) into the AatII site of plasmid pEMBLYi 22 (Baldari and Cesareni, 1985; Fig. 1). This plasmid contains the yeast URA3 gene and the DNA fragment of bacteriophage fl that permits isolation of plasmid DNA from E. coli in singlestranded form after infection with helper phage (Dente et al., 1983). Plasmid pLU was constructed by integrating the yeast L E U 2 gene (2.2-kb fragment SalI-XhoI from the plasmid YEpl3; Broach et al., 1979) into the SalI site of pLS42. The singlestranded form of plasmid pLS42 is called pEMBLYc 50, while plasmid pLU in singlestranded form is called psLU. Isolation and U V irradiation o f plasmid D N A

Fig. 1. Maps of plasmids pLS42 and pLU used in this study in single-stranded form as plasmids pEMBLYc 50 and psLU.

Single-stranded plasmids were isolated after infection of plasmid-bearing bacteria with helper

213 100--

phage, using the procedure described by Dente et al. (1983). DNA from fl helper phage constituted less than 20°7o of total DNA, as judged by agarose gel electrophoresis. UV irradiation of plasmid DNA was performed as described by White and Sedgwick (1985).

Yeast transformation and genetic analys& o f transformants Yeast cells were transformed by a modified spheroplast procedure (Zgaga et al., 1991). 40-80 ng of plasmid DNA was used for each sample, with no carrier DNA added. Under these conditions the number of transformants obtained with nonirradiated DNA was between 150 and 1000. The differences in transformation efficiency observed in different experiments seemed to depend more on protoplast preparation than on the genetic background of the strains used. For detection of leucine auxotrophs Ura + transformants were replica-plated on supplemented minimal medium lacking leucine (Sherman et al., 1986). The list of yeast strains used for transformation is given in Table 1. Results and discussion

The aim of this work was to introduce the use of yeast single-stranded replicative plasmids in the study of cellular repair processes operating on single-stranded DNA regions postulated to be the key structural intermediates in mutagenic and recombinogenic processes. As expected, UV irradiation inactivates the transforming potential of the plasmid pEMBLYc 50 much more effectively than for the equivalent double-stranded plasmid

TABLE 1 LIST OF YEAST STRAINS Strain FF18-52 FF18-66 FF565 Zl4-1b Z28-5b

Genotype

Source

M A T a , leu2-3, 112, ura3-52, ade5, C A N R F. Fabre same, but rad18.':LEU2

F. Fabre

M A Tc~, lysl, leu2, trpl, ura3-52, rev3 F. Fabre M A T a , his7, trpl, ura3-52, tad52 This work MA Tc~, lysl, ura3-52, trpl, leu2, rev3, rad52 This work

~o--

to

lO-

5-

I

I

I

[

10

25

50

75 UV

Dose (Jm -a)

Fig. 2. Transformation of different yeast strains with UVirradiated plasmid pEMBLYc 50. + , 18-52 (Rad ÷); A, 18-66 (radl8); ©, FF565 (rev3); e , Z14-1b (rad52); A, Z28-5b (rev3,

rad52).

DNA. For example, 10 J/m 2 reduced the transformation efficiency to about 50°7o (Fig. 2), whereas even a 20-fold higher dose had no effect on transformation with double-stranded plasmids of comparable size (White and Sedgwick, 1985; Keszeman-Pereira, 1990; data not shown). The main process responsible for the repair of irradiated single-stranded bacteriophages in Escherichia coli is the error-prone trans-lesion DNA synthesis (Caillet-Fauquet et al., 1977; Froehlich, 1981). The yeast mutant rev3 is defective in induced mutagenesis, and its wild-type gene product is a putative DNA polymerase involved in mutagenic repair (Morrison et al., 1989). Nevertheless, the efficiency of transformation with irradiated pEMBLYc 50 was comparable for the Rad ÷ strain and the rev3 mutant, radl8, another mutant defective in mutagenic repair, and the tad52 mutant, defective in recombinational repair, gave essentially the same result. However, the double mutant rad52 rev3 showed a significantly lower capacity

214

for the repair of UV-irradiated single-stranded plasmid (Fig. 2). In this case, the number of transformants obtained with plasmid irradiated with 10 J / m 2 was even somewhat lower than the Poisson distribution fraction of pyrimidine dimer-free D N A molecules. The expected number of dimers (0.094/plasmid m o l e c u l e / J / m 2) is calculated on the basis of the data obtained for the D N A of the bacteriophage dpX174 (Caillet-Fauquet et al., 1977). It may be slightly different for the plasmid p E M B L Y c 50 due to some specific structural feature and the difference in primary sequence (e.g., T-rich region of the centromere). Although these strains are not isogenic (with the exception of radl8 and Rad +), these results can be interpreted as evidence that in yeast the lesions in singlestranded D N A can be repaired interchangeably by mutagenic and recombinational repair processes. The source of homology for recombination may be c h r o m o s o m a l D N A (e.g., LEU2 or URA3 genes) or another plasmid if more than one molecule entered the cell during transformation. Postreplicative repair in excision-deficient strains is not affected by the rev3 mutation, and only partially impaired by the presence of the tad52 mutation (Prakash, 1981). No data for the double mutant are available. It might be that the 2 mutations have a synergistic effect on postreplicative repair, if the lesions in single-stranded D N A can be repaired by either mutagenic repair or recombination. The curves of plasmid inactivation show characteristic 'tailing' at higher doses in all strains tested (Fig. 2). This may suggest that only a fraction of t r a n s f o r m e d cells is competent for the repair of irradiated single-stranded plasmid. However, neither the synchronization of the culture with hydroxyurea nor the irradiation of cells prior to transformation (20 J / m 2) significantly influenced this response (data not shown). Although the quantity of D N A used for transformation was far from saturating, it might be that a fraction of the cell population was transformed with more than one plasmid molecule, thus having a better chance to form a Ura + colony. In order to detect possible genetic changes occurring as a consequence of repair, the LEU2 gene was

TABLE 2 G E N E T I C A N A LY S IS OF T R A N S F O R M A N T S O B T A I N E I ) W I T H U V - I R R A D I A T E D P L A S M I D psLU Strain

18-52 (Rad ~ )

UV dose ( J / m 2)

Ura ' Leu ÷

Ura~, Leu

Total

% Leu

0

251

0

251

0

25 75

515 275

5 15

520 290

/).96 5.15

FF565

0

324

0

324

0

(rev3)

75

320

4

324

1.23

introduced into plasmid pLS42 (Fig. 1). This plasmid was isolated in single-stranded form (psLU), irradiated, and used for transformation on medium lacking uracil. Transformants were replica-plated on medium lacking leucine to test for the presence of a functional LEU2 gene. The results presented in Table 2 show that UV irradiation induces the dose-dependent appearance of leucine auxotrophs among Ura + transformants. Up to 5% of Ura + transformants became Leu (15/290) in the Rad ÷ background, but only 4/324 (1.2%) in the rev3 mutant. This difference is statistically significant (P = 0.02) indicating that the product of the R E V 3 gene, which is responsible for virtually all UV-induced mutagenesis at the chromosomal level (Lemontt, 1981), also participates in mutagenesis observed on irradiated plasmid. Apparently, this is not the only pathway that can generate Ura + L e u - transformants. These might also arise by recombination between plasmid and homologous chromosomal sequences. For example, c h r o m o s o m a l mutation in the ura3 gene could be converted by the wild-type gene present on the plasmid without rescuing the plasmid molecule, so that the cell remains Leu . However, this seems unlikely, since transformation of strains carrying the ura3-52 mutation by either double- or singlestranded plasmids was never found to be achieved by gene conversion, but always by integration of the plasmid (Simon and Moore, 1987). Another possibility is the conversion of chromosomal mutations in the leu2 gene to the plasmid homologue, such that both copies of the gene present in the cell

215

become mutated. It is also possible that some other repair process may operate on irradiated singlestranded plasmids leading to changes in genetic information (e.g., by creating deletions). By sequencing the mutated plasmids, it will be possible to discover more about the mechanisms involved in the repair of UV-irradiated single-stranded plasmids.

Acknowledgements I am grateful to F. Fabre (Institut Curie, Orsay) for comments and stimulating discussions during the progress of this work and for help in preparation of the manuscript. This work was partially supported by Commission of the European Communities Joint Research Contract CI1-0528-M

(CD). References Baldari, C., and G. Cesareni (1985) Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences, Gene, 35, 27-32. Broach, J.R., J.N. Strathern and J.B. Hicks (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CANI gene, Gene, 8, 121-133. Caillet-Fauquet, P., M. Defais and M. Radman (1977) Replication in vivo of bacteriophage ~x174 single-stranded, ultraviolet light-irradiated DNA in intact and irradiated host cells, J. Mol. Biol., 117, 95-112. Dente, L., G. Cesareni and R. Cortese (1983) pEMBL: a new family of single-stranded plasmids, Nucleic Acids Res., 11, 1645-1655. di Caprio, L., and B.S. Cox (1981) DNA synthesis in UVirradiated yeast, Mutation Res., 82, 69-85. Fabre, F. (1981) Mitotic recombination and repair in relation to the cell cycle in yeast, in: H. von Wettstein, J. Friis, M. Kielland-Brandt and A. Stenderup (Eds.), Molecular Genetics in Yeast, Munksgaard, Copenhagen, pp. 399-406. Friedberg, E.C. (1988) Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae, Microbiol. Rev., 52, 70-102. Froehlich, B. (1981) Weigle reactivation of the single-stranded DNA phage fl, Mol. Gen. Genet., 184, 416-420.

Haynes, R.H., and B.A. Kunz (1981) DNA repair and mUtagenesis in yeast, in: J.N. Strathern, E.W. Jones and J.R. Broach (Eds.), The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 371-414. James, A.P., B.J. Kilbey and G.J. Prefontaine (1978) The timing of UV mutagenesis in yeast: continuing mutation in an excision-defective radl-1 strain, Mol. Gen. Genet., 165, 207-212. Keszenman-Pereira, D. (1990) Repair of UV-damaged incoming plasmid DNA in Saccharomyces cerevisiae, Photochem. Photobiol., 51, 331-342. Lemontt, J.F. (1971) Mutants of yeast defective in mutations induced by ultraviolet light, Genetics, 68, 21-31. Morrison, A., R.B. Christensen, J. Alley, A.K. Beck, E.G. Bernstine, J.F. Lemontt and C.W. Lawrence (1989) REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase, J. Bacteriol., 171, 5659-5667. Moustacchi, E. (1987) DNA repair in yeast: genetic cotrol and biological consequences, in: J. Lett (Ed.), Adv. Radiat. Biol., 13, 1-30. Orr-Weaver, T., J. Szostak and R. Rothstein (1981) Yeast transformation: a model system for the study of recombination, Proc. Natl. Acad. Sci. (U.S.A.), 78, 6354-6358. Prakash, L. (1981) Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, radl8, rev3 and rad52 mutations, Mol. Gen. Genet., 184, 471-478. Reynolds, R.J. (1987) Induction and repair of closely opposed pyrimidine dimers in Saccharomyces cerevisiae, Mutation Res., 184, 197-207. Sherman, F., G.R, Fink and J.B. Hicks (1986) Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Simon, J.R., and P.D. Moore (1987) Homologous recombination between single-stranded DNA and chromosomal genes in Saccharornyces cerevisiae, Mol. Cell. Biol., 7, 2329-2334. Singh, H., J.J. Baker and L.B. Dumas (1982) Genetic transformation of Saccharomyces cerevisiae with single-stranded DNA vectors, Gene, 20, 441-449. White, C.I., and Sedgwick (1985) The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae, Mol. Gen. Genet., 201, 99-106. Zgaga, Z., R. Chanet, M. Radman and F. Fabre (1991) Mismatch-stimulated plasmid integration in yeast, Curr. Genet., in press. Communicated by E. Moustacchi