Repair of exogenous (plasmid) DNA damaged by photoaddition of 8-methoxypsoralen in the yeast Saccharomyces cerevisiae

Mutation Research, 251 (1991) 123-131 © 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100222R

123

MUT 05016

Repair of exogenous (plasmid) DNA damaged by photoaddition of 8-methoxypsoralen in the yeast Saccharomyces cerecisiae N. Magafia-Schwencke and D. Averbeck URA 1292 du CNRS, Institut Curie, Section de Biologie, 75231 Paris Cedex 05 (France) (Received 5 February 1991) (Revision received 1 May 1991) (Accepted 6 May 1991)

Keywords: Saccharomyces cerevisiae; Survival; Plasmid; UV; 8-Methoxypsoralen; DNA repair

Summary The contribution of different repair pathways to the repair of 8-methoxypsoralen (8-MOP) plus UVA induced lesions on a centromeric plasmid (YCp50) was investigated in the yeast Saccharomyces cerevisiae using the lithium acetate transformation method. The pathways of excision-resynthesis (RAD1) and recombination (RAD52) were found to be involved in the repair of exogenous as well as of genomic DNA. Mutants in RAD6 and PS02 genes showed the same transformation efficiency with 8-MOP plus UVA treated plasmid as wild-type cells suggesting that these latter pathways involved in mutagenesis are not operating on plasmid DNA although required for the repair of 8-MOP photoadducts induced in genomic DNA. These results indicate that DNA-repair gene products may be differently involved in the repair of exogenous and endogenous DNA depending on the repair system and the nature of the DNA damage considered.

The capacity of cells to repair damaged exogenous (plasmid) DNA has been studied in bacteria (Strike et al., 1979; Mizusawa et al., 1981; Roberts and Strike, 1981; Schmid et al., 1982; Earamio et al., 1987), yeast (Dominsky and Jachymczyk, 1984; Ikai et al., 1985; White and Sedgwick, 1985, 1987a,b; Keszenman-Pereyra, 1990) and in mammalian cells (Mooibroek et al., 1984; Spivak et al., 1984; ProtiE-Sablji6 and Kraemer, 1985; Van Duin et al., 1985; Nairn et al., 1988). The use of exoge-

Correspondence: Dr. D. Averbeck, URA 1292 du CNRS, Institut Curie, Section de Biologie, 26 rue d'Ulm, 75231 Paris Cedex 05 (France).

nous DNA for repair studies has the obvious advantage that defined DNA lesions can be introduced in vitro, without damaging cell components other than DNA. After introduction of damaged plasmid into competent cells by transformation, the in vivo repair of the DNA lesions by cellular enzymes can be followed by determining the expression of selectable genetic markers from the plasmid in outgrowing colonies. In other ~vords, the survival of transformants reflects the repair of plasmid DNA. The contribution of cellular repair pathways to plasmid repair can be assessed using defined repair-defective mutants as host cells. This is particularly of interest in eukaryotic cells for which the enzymatic steps for the repair of

124

specific lesions in DNA need to be elucidated. Genetically well characterized repair mutants (rad) exist in the unicellular eukaryotic yeast Saccharomyces cerevisiae, which makes it an ideal organism to study DNA repair at the molecular level. Three main repair pathways can be distinguished: the excision-resynthesis (RAD3 type), the mutagenic (RAD6 type) and the strand-break and recombinational (RAD52 type) repair pathways (see for review Moustacchi, 1987; Friedberg, 1988). Studies on the repair of plasmid UV-irradiated in vivo (McCready and Cox, 1980; Smerdon et al., 1990) and in vitro prior to transformation of wild-type and DNA repair-deficient mutants (Dominsky and Jachymczyk, 1984; Ikai et al., 1985; White and Sedgwick, 1985, 1987a,b; Keszenman-Pereyra, 1990) have been reported in yeast. However, controversial results were obtained concerning the involvement of excision-resynthesis gene products (i.e., RAD1, 2, 3, 4, 7 and 14) in plasmid repair. The reasons for the differences observed are not clear and more work is needed to clarify especially the importance of the mutant alleles and the different strains used. As seen by the survival of UV-treated plasmid after transformation, the mutants belonging to the RAD52 epistasis group were slightly affected in the repair of UV-damaged plasmid DNA as well as in the repair of chromosomal DNA (White and Sedgwick, 1985; Keszenman-Pereyra, 1990). The same authors reported that rad6 defective in mutagenic repair showed little effect on the repair of UV-damaged exogenous DNA, but pronounced effects on the repair of genomic DNA and on cell survival after UV treatment. In view of these results, we were interested to examine the contribution of the repair pathways of the yeast S. cerevisiae to the repair of other types of lesions in exogenous compared to chromosomal DNA. In the presence of near-ultraviolet or UVA (320-380 nm) radiation, furocoumarins such as 8-methoxypsoralen (8-MOP) are able to induce C4-cyclobutane additions to pyrimidines in DNA, i.e., monoadducts and interstrand cross-links (Ben-Hur and Song, 1984; Cimino et al., 1985). In yeast, the 3 main repair pathways previously described, as well as specific pathways (PSO2-type) for the repair of damage

TABLE 1 STRAINS OF Saccharomyces cerevisiae Strain

Genotype

FF1852

MATa leu2-3 trpl-289 ura3-52 ade5 canl RAD MA Ta leu2-3 trpl-289 ura3-52 ade5 radl-1 MA Ta leu2-3 trp l-289 ura3-52 his 7 rad52 MATa leu2-3 trpl-289 ura3-52 his7 radl-1 rad52 MATa leu2-3 trpl-289 rad3-52 his3A1 horn3 rad6-1 MA Ta leu2-3 trpl-289 ura3-52 his1 ade5 pso2-1

ZZ72 ZZ43 ZZ26-2C YMKZ CC-6B

Source F. Fabre Z. Zgaga Z. Zgaga Z. Zgaga M. Kupiec C. CassierChauvat

induced by cross-linking agents, are known to operate on chromosomal DNA damage photoinduced by furocoumarins (see for review Moustacchi, 1987; Friedberg, 1988; Averbeck, 1989). In the present paper, we investigate the capacity of the different pathways to participate in the repair of 8-MOP plus UVA induced damage in plasmid DNA. Materials and methods

Strains We employed the Escherichia coli strain HB 101 for the amplification of plasmids. The genotypes of the yeast strains used are specified in Table 1. The strains were kindly provided by Dr. F. Fabre (Institut Curie-Biologie, Orsay, France).

Media and buffers Yeast cells were grown in liquid YPD medium (1% yeast extract (Difco), 2% Bacto peptone (Difco) and 2% glucose). Transformants were plated on solid minimal medium (1% Bacto yeast nitrogen base without amino acids, 2% glucose, 2.5% Bacto agar (Difco)) supplemented with the amino acids and nitrogen bases appropriate for each strain. Bacteria were grown in Luria-Bertani medium (Maniatis et al., 1982) supplemented with 50 /zg/ml ampicillin. TE consisted of 10 mM Tris-HC1, 1 mM EDTA, pH 8. The transformation buffer was 0.1 M lithium acetate in TE, pH 7.5.

125

Plasmids The yeast shuttle vector YCp50 has 8040 bp, is derived from pBR322 and contains the yeast autonomous replication sequence ARS1, the URA3 gene and the centromeric sequence of chromosome IV (CEN4) (Johnston and Davis, 1984). YEpl3 contains the origin of replication from 2 /~ and the LEU2 gene (Broach et al., 1979). The plasmid YRp7 is derived from pBR322 and contains the TRP1 gene and an ARS sequence (Tschumper and Carbon, 1980). The plasmids are prepared by amplification in E. coli HB 101. Clear lysates are purified by centrifugation in CsCl isopycnic gradients.

UV 250

\\\

_A

~

Treatment of cells Ceils were grown in YPD to exponential phase (1-2 × 10 7 cells/ml). They were washed and divided into 2 parts, one for transformation and the other, for cell survival, was resuspended in water at 3 × 10 6 cells/ml and incubated with 8-MOP (50 /~M) at 4 ° C for 20 min (Magafia-Schwencke et al., 1982). Then controls were taken and other cell samples exposed to different doses of UVA (365 nm) radiation at a fluence rate of 10 j / m 2 / s and plated on minimal medium with appropriate supplements. Survivors were scored by counting outgrowing colonies after 5 days of growth. For exposure to UVA, a HPW 125 Philips lamp (peak emission at 365 nm) was used in connection with a digital radiometer J-260 (Ultraviolet Products, San Gabriel, CA, U.S.A.) (Averbeck, 1985). Treatment of DNA with 8-MOP Plasmid DNA (20 ng/~,l in TE) was incubated with 8-MOP at 50 /,M for 20 rain at 4 ° C and irradiated with different doses of UVA of 365 nm (HPW 125 Philips lamp) at a fluence rate of 10 j / m 2 / s . After irradiation, DNA was precipitated with ethanol, washed in ethanol 70%, dissolved in TE and reprecipitated and washed once more. It was then dissolved in TE at 200 n g / > l and used for transformation. Treatment of DNA with UV of 254 nm Plasmid DNA (20 ng//~l in TE) was irradiated at 10 J / m Z / s with a low-pressure germicidal UV lamp (254 nm). At different intervals, aliquots are taken and used to transform yeast.

500

dose

( K J m -2)

250

500

wf

lO-

0

254

250

I1~II

500

w,

\

\

.

•

\

\ ~

d.~

10"2

co

•

10 -3

YCp 50 I

I

YEp13 I

I

YRp7 I

I

]Fig. 1. Survival curves of UV-irradiated D N A of plasmid YCp50 (panel A), YEpl3 (panel B) and YRp7 (panel C) transformed into the wild-type and the excision-deficient radl-1 strain of Saccharomyces cerevisiae.

Yeast transformation This was performed with the lithium acetate method (Ito et al., 1983) with minor modifications, or the protoplast method (Hinnen et al., 1978). The transformed ceils were plated on minimal medium with the necessary supplements. Transformant colonies were scored after 7 days of growth at 30°C. Results

Transformation efficiency of 254 nm UV-damaged plasmids In order to test the performance of the yeast transformation system used, the transformation efficiencies of the UV-irradiated centr~)meric plasmid YCp50 were determined in wild-type RAD + cells and radl mutant cells and the results compared to those of the plasmid YEpl3 derived from the natural yeast plasmid 2 /, and the multicopy plasmid YRp7. Fig. 1 shows the survival of the UV-irradiated

126 UVA d o s e 1

2

3

4

5

6

( KJ rn I

-2 )

2

3

4

A

1

5

-T-'--B

°

10-2i

• ad52

F_

pso

/°

L

(n 10- 3

_

• ~ r (~dl ~

d6

•

10- 4

Fig. 2. Survivalcurves obtained after treatment with 8-MOP plus UVA (365 nm) radiation of haploid exponentiallygrowing cells of Saccharomyces cereuisiae. (A) Wild-type, rad52, radl-1 and radl rad52 cells. (B) Wild-type, rad6 and pso2 cells. plasmids YCp50, Y E p l 3 and YRp7 after transformation of ReiD + and radl protoplasts by measuring transformability to uracil, leucine and tryptophan prototrophy, respectively. Similar results were obtained by transformation of cells with the lithium acetate method (data not shown). Thus, both transformation methods appear to be suitable for repair studies in yeast with 8-MOP plus U V A damaged plasmids. For the different plasmids used, survival in the radl strain was approximately the same following UV exposure (Fig. 1) and the UV response was exponential. Survival of UV-irradiated plasmid in the corresponding wild-type RAD + strain yielded shouldered dose-response curves. The response of UV-irradiated plasmids YCp50 and YRp7 in the RAD + strain was approximately the same whereas UV-irradiated plasmid Y E p l 3 gave a higher survival. A comparison of the doses for 37% plasmid

survival (Table 2) indicates that the UV-irradiated plasmids YCp50 and YRp7 are by a factor of approximately 3 more sensitive in terms of survival in the radl mutant than in the wild-type host. Because of the higher U V survival of Y E p l 3 plasmid in wild-type cells, the sensitivity factor (SF) for radl mutant, i.e., the ratio of the D37 of the wild type to the D37 of the mutant radl, amounts to 5.1. Survival of 8-MOP plus UVA treated plasmid YCp50 in transformable wild-type and mutant strains In order to allow comparisons in the repair of plasmid and chromosomal D N A in yeast cells of the same physiological conditions, we measured cell transformation after treatment with 8-MOP and U V A using the same cultures as for the determination of cell survival. Fig. 2A,B shows

127 plasmid is decreased in the mutants radl, rad52 and in the double mutant radl rad52, in comparison to that in wild-type cells (Fig. 3A), whereas it is approximately the same as in the wild-type in the mutants rad6 and pso2 (Fig. 3B). The parameters for plasmid survival are given in Table 3B. The sensitivity factors for plasmid survival (Table 3B) are generally smaller than those for cell survival (Table 3A). Sensitivity increases when comparing the response of the double mutant radl rad52 with that of the single mutants radl and tad52. As seen above (Fig. 2B), tad6 and pso2 mutants are sensitive to the lethal effects of 8-MOP plus U V A in comparison to the wild-type cells. In contrast, survival of 8-MOP plus U V A treated plasmid in rad6 and pso2 cells is approximately the same as in wild-type cells (Fig. 3B), suggesting

survival curves obtained for the wild-type strain RAD + and for the mutants radl, rad52, and radl rad52 as well as for the mutant strains rad6 and pso2. As reported previously (Averbeck and Moustacchi, 1975; Henriques and Moustacchi, 1980a,b), the excision-defective mutant radl-1, the mutant rad52 deficient in recombination, the mutant rad6 deficient in mutation, the mutant pso2 defective in cross-link repair and the double mutant radl rad52 are sensitive to 8-MOP plus U V A treatment. The parameters of the survival curves are given in Table 3A. Transformable haploid yeast strains carrying the wild-type, radl-1, rad52, radl-1 rad52, rad6-1 and pso2-1 alleles were transformed to uracil prototrophy by the centromeric plasmid YCp50 treated with 8-MOP plus UVA. Fig. 3A,B shows that the survival of 8-MOP plus U V A treated

UVA d o s e 1

2

3

4

5

(KJ m -2)

6

1

2

3

4

5

6

I wf

&

~

PS° 2

10-2

L. J

Pad

I - r

10-3

10-41

~1~

I

t

I

I

I

I

--

I

I

I

I

I

I

Fig. 3. Survivalcurves of plasmid DNA (YCp50) previouslytreated with 8-MOP plus UVA (365 nm) radiation and transformed into wild-type and repair-deficient strains of Saccharomycescerevisiae.(A) Results obtained with wild-type, tad52, radl and radl rad52 cells. (B) Results obtained with wild-type, tad6 and pso2 cells.

128 TABLE 2 PARAMETERS FOR UV (254 nm)-IRRADIATED PLASMIDS IN TRANSFORMABLE WILD-TYPE AND radl MUTANT STRAINS OF Saccharomyces cerevisiae Strain

Shape of curve (Dq in J/m 2)

D37 (J/m 2)

Sensitivity factor

shouldered(120) exp (0)

290 85

3.4

shouldered(60) exp (0)

460 90

5.1

shouldered(60) exp (0)

335 110

3

YCp50 survival

Wild-type radl YEpl3 survival

Wild-type radl YRp 7 survival

Wild-type radl

Dq, quasi-threshold dose defining the extent of the shoulder; D37, dose for 37% survival; Sensitivity factor, ratio of D37 wild-type to mutant. that the R A D 6 and P S 0 2 proteins are not involved in the repair of 8-MOP plus U V A induced lesions on exogenous plasmid DNA.

Discussion In the present paper, we examined the repair of 8-MOP plus U V A damaged centromeric plasmid D N A (YCp50) in highly transformable haploid strains of the yeast Saccharomyces cerevisiae. As pointed out by Keszenman-Pereyra (1990), during the procedure used for whole-cell transformation, only a single plasmid D N A molecule is established per cell. Furthermore, according to

White and Sedgwick (1985), the uptake of UVdamaged plasmid is not affected by fluences up to 1700 J / m e. Thus, the survival of incoming plasmid D N A can be correlated with the inhibitory action of UV lesions on replication a n d / o r the expression of selectable marker genes. This can be expected to be true also for 8-MOP plus U V A damaged plasmid DNA. In a series of control experiments performed with UV-irradiated plasmid DNAs, the protoplast transformation procedure as well as the lithium acetate method gave results (Fig. 1 and Table 2) that are consistent with those obtained by other authors (White and Sedgwick, 1985; Keszenman-Pereyra, 1990). For instance, the survival of UV-damaged plasmid YCp50 in the radl mutant is decreased by a factor of approximately 3.4 in comparison to that in wild-type cells and is in the same range as that reported by Keszenman-Pereyra (1990). The 3 main repair pathways known in yeast (excision-resynthesis, mutagenic and recombinogenic) as well as the P S O 2 - d e p e n d e n t pathway (specifically defective in the repair of cross-linked D N A ) have been shown to be implicated in the repair of psoralen-induced damage (see for review Moustacchi, 1987; Friedberg, 1988) in genomic DNA. The survival data obtained after treatment with 8-MOP plus U V A using cells in the same conditions as in the transformation studies confirm the respective importance of the different repair pathways (Fig. 2A,B). When comparing the parameters of the survival curves with those obtained for plasmid survival interesting differences and similarities are

TABLE 3 PARAMETERS FOR THE SURVIVALOF 8-MOP PLUS UVA TREATED PLASMID(YCp50) AND HOST CELLS Strain

wild-type rad52 radl tad1 rad52 rad6 pso2

(A) Host cell survival

(B) Plasmid survival

Dq (kJ/m 2)

D37 (kJ/m 2)

Sensitivity factor

Dq (kJ/m 2)

D37 (kJ/m 2)

Sensitivity factor

0.75 0.39

1.54 0.65 0.30 0.15 0.84 1.14

2.4 5.1 10.3 1.8 1.4

0.55 0.15 0 0 0.5 0.65

2.7 1.25 0.95 0.85 2.55 2.85

2.2 2.8 3.2 1.06 0.96

0 0

0.54 1.00

129

noticed (Table 3). The sensitivity factors for the radl-1 strain and the rad52-1 strain are in the same range for both biological endpoints, suggesting that the excision-resynthesis and the DNA strand-break and recombinational pathways contribute approximately to the same extent to the repair of 8-MOP plus UVA induced lesions in genomic and in exogenous plasmid DNA. The sensitivity factor obtained in the radl rad52 double mutant for cell survival (Table 3A) is higher than that for plasmid survival (Table 3B), indicating that the interaction of the 2 pathways is more important for genomic DNA than for plasrnid DNA repair. Plasmid survival was approximately the same in the rad6 mutant and pso2 mutant strains as in wild-type cells (Fig. 3B), whereas it was strongly reduced in radl and rad52 ceils and in the radl rad52 double mutant (Fig. 3A). The results obtained on plasmid survival are consistent with the hypothesis that the RAD6 and PS02 products are not operating on exogenous DNA carrying 8-MOP plus UVA induced lesions. This finding is especially surprising because the RAD6- as well as the PSO2-dependent pathways are known to play an important role in the repair of 8-MOP plus UVA induced lesions in genomic DNA, as demonstrated by the fact that rad6 and pso2 mutants are sensitive to 8-MOP plus UVA treatment (Fig. 2B). The explanation for these results may lie in the fact that the protein encoded by the RAD6 gene is a ubiquitin-conjugating enzyme implicated in the ubiquitination of histones and other proteins, possibly inducing chromatin rearrangements a n d / o r affecting the accessibility of other repair enzymes to DNA lesions (Jentsch et al., 1987). During the first steps of transformation, the entering plasmid is likely to be naked a n d / o r partially folded into chromatin (Smerdon et al., 1990). Consequently, the accessibility of DNA lesions on plasmid DNA is expected to differ from that of lesions in genomic DNA, and the repair of plasmid DNA damage may not require the action of the RAD6 gene product. The pso2-1 mutation in yeast is known to confer high sensitivity to the lethal effects of bifunctional agents inducing DNA interstrand cross-links (Cassier et al., 1980; Henriques and

Moustacchi, 1980a; Cassier and Moustacchi, 1981; Saeki et al., 1983). The fact that the survival of 8-MOP plus UVA treated plasmid in the pso2-1 mutant strain is the same as in the wild-type host implies that the PS02 protein normally required for the repair of cross-links in genomic DNA is not needed for the repair of these lesions in plasmid DNA. In vivo, biochemical studies on the fate of 8-MOP plus UVA induced cross-links in yeast (Magafia-Schwencke et al., 1982) have shown that the pso2-1 mutant is able to incise 8-MOP plus UVA induced interstrand cross-links and to produce single- and double-strand breaks as efficiently as the wild-type during a period of post-treatment incubation. However, the pso2-1 mutant failed to perform the DNA strand-resealing step (Magafia-Schwencke et al., 1982). So far, no information is available on the activity of the PS02 protein, but from its differential requirement for the repair of lesions in genomic DNA and plasmid DNA, it is tempting to assume that its action may depend on chromatin structure. In conclusion, measurements of plasmid DNA repair are useful to study the contribution of repair pathways. However, our results indicate that DNA-repair gene products may be differently involved in the repair of exogenous and endogenous DNA depending on the nature of the repair system and the type of DNA damage considered. Acknowledgements Financial support by the Centre National de la Recherche Scientifique (CNRS) and the Ligue Nationale Fran§aise contre le Cancer is gratefully acknowledged. Wc thank Madame Dominique Chardonnieras for excellent assistance. We are very much indebted to Dr. Francis Fabrc and co-workers for the kind gift of repair-deficient transformation-competent yeast strains and plasmids. We thank Dr. E. Moustacchi for her critical review of the manuscript and helpful suggestions. References Averbeck, D. (1985) Relationship between lesions photoinduced by mono- and bifunctional furocoumarins in DNA and genotoxic effects in diploid yeast, Mutation Res., 151, 217-233. Averbeck, D. (1989) Yearly review: Recent advances in pso-

130 ralen phototoxicity mechanism, Photochem. Photobiol., 50, 859-882. Averbeck, D., and E. Moustacchi (1975) 8-Methoxypsoralen plus 365 nm light effects and repair in yeast, Biochim. Biophys. Acta, 395, 393-404. Ben-Hut, E., and P.S. Song (1984) The photochemistry and photohiology of furocoumarins (psoralens), in: Advances in Radiation Biology, Vol. 11, Academic Press, New York, pp. 131-171. Broach, J.R., J.N. Strathern and J.B. Hicks (1979) Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene, Gene, 8, 121-133. Cassier, C., and E. Moustacchi (1981) Mutagenesis induced by mono- and bifunctional alkylating agents in yeast mutants sensitive to photo-addition of furocoumarins (pso), Mutation Res., 84, 37-47. Cassier, C., R. Chanet, J.A.P. Henriques and E. Moustacchi (1980) The effects of three PSO genes on induced mutagenesis: a novel class of mutationally defective yeast, Genetics, 96, 841-857. Cimino, G.D., H.B. Gamper, S.T. Isaacs and J.E. Hearst (1985) Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry and biochemistry, Annu. Rev. Biochem., 54, 1151-1193. Dominski, Z. and W.J. Jachymczyk (1984) Repair of UVirradiated plasmid DNA in a Saccharomyces cerevisiae tad3 mutant deficient in excision-repair of pyrimidine dimers, Mol. Gen. Genet., 193, 167-171. Friedberg, E.C. (1988) Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae, Microbiol. Rev., 52, 70102. Henriques, J.A.P., and E. Moustacchi (1980a) Isolation and characterization of pso mutants sensitive to photoaddition of psoralen derivatives in Saccharomyces cerevisiae, Genetics, 95, 273-288. Henriques, J.A.P., and E. Moustacchi (1980b) Sensitivity to photoaddition of mono- and bifunctional furocoumarins of X-ray-sensitive mutants of Saccharomyces cerevisiae, Photochem. Photobiol., 31,555-562. Hinnen, A., J.B. Hicks and G.R. Fink (1978) Transformation of yeast, Proc. Natl. Acad. Sci. (U.S.A.), 75, 1929-1933. Ikai, K., K. Tano, T. Ohnishi and K. Nozu (1985) Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerecisiae, Photochem. Photobiol., 42, 179-181. Ito, H., Y. Fukuda, K. Murata and A. Kimura (1983) Transformation of intact yeast cells treated with alkali cations, J. Bacteriol., 153, 163-168. Jentsch, S., J.P. McGrath and A. Varshavsky (1987) The yeast DNA repair gene RAD6 encodes an ubiquitin-conjugating enzyme, Nature (London), 239, 131-134. Johnston, M., and R.W. Davis (1984) Sequences that regulate the divergent GALI-GALIO promoter in Saccharomyces cerevisiae, Mol. Cell. Biol., 4, 1440-1448. Keszenman-Pereyra, J.R. (1990) Repair of UV-damaged incoming plasmid DNA in Saccharomyces cereL,isiae, Photochem. Photobiol., 51,331-342. McCready, S.J., and B.S. Cox (1980) Repair of 2 ~ m plasmid

DNA in Saccharomyces cerevisiae, Curr. Genet., 2, 207210. Magafia-Schwencke, N., J.A.P. Henriques, R. Chanet and E. Moustacchi (1982) The fate of 8-methoxypsoralen photoinduced cross-links in nuclear and mitochondrial yeast DNA: comparison of wild-type and repair deficient strains, Proc. Natl. Acad. Sci. (U.S.A.), 79, 1722-1726. Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mizusawa, H., C. Lee and T. Kakefuda (1981) Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide in Escherichia coli, Mutation Res., 82, 4557. Mooihroek, H., B. de Jong and G. Venema (1984) Repair of UV damage in plasmid DNA by human fibroblasts, Mol. Gen. Genet., 195, 175-179. Moustacchi, E. (1987) DNA repair in yeast: genetic control and biological consequences, Adv. Radiat. Res., 13, 1-30. Nairn, R.S., R.M. Humphrey and G.M. Adair (1988) Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids, Int. J. Radiat. Biol., 53, 249-260. Paramio, J.M., C. Bauluz and R. de Vidania (1987) Lethal and mutagenic effects of 8-methoxy-psoralen-induced lesions on plasmid DNA, Mutation Res., 176, 21-28. Proti6-Sablji6, M., and K.H. Kraemer (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells, Proc. Natl. Acad. Sci. (U.S.A.), 82, 6622-6626. Roberts, R.J., and P. Strike (1981) Efficiency of Escherichia coli repair processes on UV-damaged transforming plasmid DNA, Plasmid, 5, 213-220. Saeki, T., C. Cassier and E. Moustacchi (1983) Induction in Saccharomyces cerevisiae of mitotic recombination by mono- and bifunctional agents: comparison of the pso2-1 and tad52 repair deficient mutants to the wild-type, Mol. Gen. Genet., 190, 255-264. Schmid, S.E., M.P. Daune and R.P.P. Fuchs (1982) Repair and mutagenesis of plasmid DNA modified by ultraviolet irradiation on N-acetoxy-N-acetylaminofluorene, Proc. Natl. Acad. Sci. (U.S.A.), 79, 4133-4137. Smerdon, M.J., J. Bedoyan and F. Thoma (1990) DNA repair in a small yeast plasmid folded into chromatin, Nucleic Acids Res., 18, 2045-2051. Spivak, G., A.K. Ganesan and P.C. Hanawalt (1984) Enhanced transformation of human cells by UV-irradiated pSV2 plasmids, Mol. Cell. Biol., 4, 1169-1171. Strike, P., G.O. Humphreys and R.J. Roberts (1979) Nature of transforming deoxyribonucleic acid in calcium-treated Escherichia coli, J. Bacteriol., 138, 1033-1035. Tschumper, G., and J. Carbon (1980) Sequence of yeast DNA fragment containing a chromosomal replicator and the TRP1 gene, Gene, 10, 157-166. Van Duin, M., A. Westerveld and J.H.J. Hoeijmakers (1985) UV stimulation of DNA-mediated transformation of human cells, Mol. Cell. Biol., 5, 734-741.

131 White, C.I., and S.G. Sedgwick (1985) The use of plasmid DNA to probe DNA repair functions in the yeast Saccharomyces cerevisiae, Mol. Gen. Genet., 201, 99-106. White, C.I., and S.G. Sedgwick (1987a) Induced cellular resistance to ultraviolet light in Saccharomyces cerevisiae is not accompanied by increased repair of plasmid DNA, Curr. Genet., 11,321-326.

White, C.I., and S.G. Sedgwick (1987b) Repair of UVirradiated plasmid DNA in Saccharomyces cerevisiae. Inability to complement mutational defects in excision repair by in vitro treatment with Micrococcus luteus UV endonuclease, Mutation Res., 183, 161-167.