Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and

Vol. 4, No. 2MOLECULAR AND CELLULAR BIOLOGY, Feb. 1984, p. 290-2950270-7306/84/020290-06$02.00/0Copyright C 1984, American Society for Microbiology

Saccharomyces cerevisiae RAD2 Gene: Isolation, Subcloning, andPartial Characterization

LOUIE NAUMOVSKI AND ERROL C. FRIEDBERG*Laboratory of Experimental Oncology, Department of Pathology, Stanford University, Stanford, California 94305

Received 6 September 1983/Accepted 9 November 1983

A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity ofrad2-1, rad2-2, and rad24 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomiclibrary. Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmidcontaining a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at thechromosomal rad2 gene. We therefore conclude that this plasmid contains the RAD2 gene. The 9.3-kilobasepair fragment was partially digested with Sau3A and cloned into a multicopy yeast vector designed for easyretrieval of Sau3A inserts. The smallest subclone that retains the RAD2 gene is 4.5 kilobase pairs. Thisfragment was partially digested with Sau3A and cloned into an integrating plasmid. These plasmids wereisolated and integrated into a heterozygous rad2IRAD2 strain. Plasmids containing internal fragments of theRAD2 gene were identified because they yielded UV-sensitive transformants due to disruption of the RAD2gene. Sporulation of diploids transformed with integrating plasmids containing internal fragments of RAD2gave rise to four viable haploids per tetrad, indicating that unlike the RAD3 gene of S. cerevisiae, the RAD2gene is not essential for the viability of haploid cells under normal growth conditions. Measurements of theRNA transcript by RNA-DNA hybridization with the internal fragment as the probe indicate a size of -3.2kilobases.

Previous studies have established that the RADI, RAD2,RAD3, RAD4, and RADIO genes of the yeast Saccharomy-ces cerevisiae are required for the incision of DNA duringthe excision repair of pyrimidine dimers produced by UVradiation (11, 16). To investigate the function of the productsencoded by these genes, we have isolated recombinantplasmids that complement the UV sensitivity of mutantsdefective in each of the RAD loci mentioned above. We havereported elsewhere that plasmid pNF3000 contains the yeastRAD3 gene (6). In this paper we describe the isolation of aplasmid designated pNF2000 and demonstrate that it con-tains the yeast RAD2 gene. We also describe the subcloningand partial characterization of this gene.

MATERIALS AND METHODSYeast and bacterial strains and plasmids. The S. cerevisiae

yeast and bacterial strains used in this study are shown inTable 1. Haploid rad- strains carrying the ura3-52 mutationwere constructed by mating rad mutants to strain SX46A(ura3-52), sporulating the diploids, and isolating the appro-priate haploids. Strains radi-Ji, rad2-1, rad3-2, rad4-3, andradl4-1 were obtained from the Yeast Genetic Stock Center,Berkeley, Calif. The rad2-2 and rad24 strains were providedby R. Reynolds, School of Public Health, Harvard Universi-ty, Boston, Mass. The recombinant yeast DNA plasmid poolwas constructed by Carlson and Botstein (1). The plasmidsYRp17 and YRp18 contain the yeast URA3 gene as well asyeast autonomously replicating sequences and were ob-tained from R. Davis, Stanford University, Stanford, Calif.The integrating plasmid YIp5 also contains the yeast URA3gene but does not have a replication origin that is active in S.cerevisiae (12). For subcloning partial Sau3A digests ofRAD2, the vectors pNF2 and pNF3 were used (7).

Culture media. Escherichia coli HB101 was grown in L

* Corresponding author.

broth, supplemented with ampicillin (50 ,ug/ml) when appro-priate. Yeast minimal medium consisted of 0.17% yeastnitrogen base, 0.5% ammonium sulfate, and 2% dextrose.This medium was supplemented with adenine (30 ,ug/ml),tryptophan (30 p.g/ml), histidine (30 ,ug/ml), and uracil 20,ug/ml) as required. Complete medium (YPD) contained 1%yeast extract, 2% peptone, and 2% dextrose. Sporulationplates contained 0.965% potassium acetate, 0.25% yeastextract, 0.2% dextrose, and 2% agar. For transformationwith plasmid DNA, Glusulase-treated yeast cells were platedin regeneration agar consisting of supplemented minimalmedium plus 1.0 M sorbitol in 3% agar.

Preparation of DNA. Rapid lysates of S. cerevisiae and ofE. coli were prepared as described previously (6). Large-scale isolation of plasmid DNA from E. coli and purificationin cesium chloride-ethidium bromide gradients was carriedout as described in Davis et al. (2), except that the ethidiumbromide concentration was 200 ,ug/ml.

Transformation of cells with DNA. Transformation of E.coli with plasmid DNA was carried out according to themethod of Okayama and Berg (10). Transformation of yeastcells was performed according to the procedure of Hinnen(3), with modification as previously described (6).

Characterization of DNA. Restriction enzymes were pur-chased from Bethesda Research Laboratories, Bethesda,Md., and from New England Biolabs, Boston, Mass., andwere used according to directions in Davis et al. (2). Calfalkaline phosphatase was obtained from Boerhinger Mann-heim Biochemicals, Indianapolis, Ind. Gel electrophoresis ofDNA, 32P-labeling of DNA by nick translation, and DNA-DNA hybridization were performed as described by Davis etal. (2).Measurement of UV survival of yeast strains. Quantitative

UV survival of transformed S. cerevisiae strains was per-formed as described previously (6).

General subcloning procedures. For all subcloning ofDNAfragments, recipient vectors were linearized with appropri-ate restriction enzymes, and the DNA was treated with calf

290

S. CEREVISIAE RAD2 GENE 291

TABLE 1. Bacterial and yeast strains

Strain Relevant genotype Source

E. coliHB101 J. Rubenstein, Stanford UniversityLE392 J. Rine, Stanford UniversitySR73 uirA6 recA13 S. Lloyd, Stanford UniversitySR57 uvrC34 recA56 N. Sargentini, Stanford UniversitySR58 uv'rB5 recA56 N. Sargentini, Stanford University

S. cerevisiae

SX46A MATa ura3-52 trpl-289 his3-832 ade2 J.Rine, Stanford UniversityXR270-34C MATa his4-580(Am) trpl(Am) tyr2(0c) ade2(0c) J. Rine, Stanford UniversityLN2-115 MATa ura3-52 trpl-289 his3-832 ade2 rad2-1 This study

MA Tot ura3-52 TRP HIS ade2 RAD2YM-197 MATa ura3-52 lvs2-801(Am) ade2-101(Oc) GAL SUC2 M. Johnston, Stanford University

MATa ura3-52 xvs2-801(Am) ade2-101(Oc) GAL SUC2LN1-11I1 MATot radl-Il ura3-52 This studyLN2-1I1 MATax rad2-1 ura3-52 This studyLN2-213 MATot rad2-2 ura3-52 trpl-289 This studyLN2-4110 MATa rad24 ura3-52 his3-832 ade2 This studyLN3-214 MATa rad3-2 itra3-52 trpl-289 ade2 This studyLN4-315 MATa rad4-3 ura3-52 trpl-289 This studyLN14-111 MATot radl4-1 ura3-52 trpl-289 This study

alkaline phosphatase to prevent self-ligation. Enzymes wereinactivated and DNA was recovered by phenol extraction,followed by extraction with ether and precipitation withethanol. Ligations were carried out by using T4 DNA ligaseat 4°C for 16 h.

Subcloning of pNF2000 in the vector pNF2. The 9.3-kilobase pair (kbp) HindlIl fragment of pNF2000 was isolat-ed by using sodium iodide and glass beads (15) and wasincubated with the Sau3A enzyme to generate a partialdigest. Fragments were ligated into the BamHI site of theYEp24-derived vector pNF2 (7). The mixture was trans-formed into E. coli, and cells were grown overnight in Lbroth in the presence of ampicillin. Plasmid DNA from thispool was isolated and purified by centrifugation in cesiumchloride-ethidium bromide gradients and then used to trans-form a ura3-52 rad24 yeast strain. Yeast colonies resistantto UV radiation were identified and subcultured on minimalmedium in the absence of uracil to select for coloniescontaining only a single species of plasmid. DNA wasisolated from rapid lysates and propagated in E. coli. Theplasmids isolated in this way were mapped by digestion withSall enzyme to ascertain the size of the DNA insert and withEcoRI, XhoI, and KpnI to determine the location of thesubcloned fragment relative to the 9.3-kbp HindIll fragment.

Isolation and identification of an internal fragment of theRAD2 gene. The 4.5-kbp subcloned fragment containing theRAD2 gene was partially digested with Sau3A, and frag-ments were ligated into the BamHI site of pNF3, an integrat-ing vector (7). This pool was transformed into E. coli, andindividual colonies were screened for plasmids with DNAinserts. Three plasmids with inserts of 2.2, 2.2, and 1.0 kbpwere isolated, and the position of each relative to the 4.5-kbpfragment was mapped by restriction analysis. The identifica-tion of those plasmids containing internal fragments of RAD2was determined by transformation of a RAD21rad2-1 hetero-zygous diploid and examining the RAD phenotype of thetransformants.

Identification of RAD2 gene transcript. A 200-ml overnightculture of strain S288C was grown at 30°C with vigorousshaking to an optical density at 600 nm of -1.0. RNA wasextracted by the procedure of R. Elder as described in

reference 4. Total cellular RNA was treated with glyoxal asdescribed (5), and the equivalent of 50 to 100 ,ug of RNA wasloaded onto a 1% horizontal agarose gel. Electrophoresiswas in 10 mM sodium phosphate buffer at 100 V for 4 h.RNA was transferred to nitrocellulose paper and treated asdescribed (14), except that 10% sodium dextran sulfate wasincluded in the prehybridization and hybridization buffer.The DNA probe used for hybridization contained the URA3gene and an internal fragment of the RAD2 gene and waslabeled by nick translation to a specific radioactivity of -5 x107 cpm/,lg. After hybridization, filters were washed, andthe presence and size of RNA bands were determined byautoradiography.

RESULTSIsolation of plasmid pNF2000. A rad2-2 ura3-52 mutant of

S. cerev'isiae was transformed with a Sau3A yeast gene poolconstructed by Carlson and Botstein (1). After the screeningof about 2,000 transformants, one colony was identified asUV resistant both by the replica plating test and the moresensitive streak test previously described (6). After beingsubcultured in minimal medium, a number of colonies wereindividually tested for linkage of the URA3 gene and the UV-resistance determinant. In all cases strict cosegregation was

.i §_ \

\ * 00,

pNF2000

rod 1- IlrQd 2-4rod 3-1rod4-4rod 14 - 1RAD+

.~~~~~-1

\ \ :~~w:

YE p24FIG. 1. Plasmid pNF2000 confers enhanced UV resistance to the

rad24 mutant but not to other rad mutants. Experimental details ofthe streak test are described in the text.

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292 NAUMOVSKI AND FRIEDBERG

observed, i.e., loss of the URA3 marker was accompaniedby loss of the UV-resistance determinant, indicating theirpresence on a single plasmid. Plasmid DNA isolated from asubculture of the UV-resistant colony was propagated in E.coli and showed the presence of a single plasmid by restric-tion analysis. This plasmid (pNF2000) transforms rad2-1,rad2-2, and rad24 mutants of S. cerevisiae to enhanced UVresistance, but it has no effect on the UV sensitivity of radl-11, rad3-1, rad44, or radl4-1 mutants (Fig. 1). Quantitativesurvival curves for rad2-2 and rad24 transformed withpNF2000 showed that the latter plasmid confers normallevels of UV resistance to the mutants relative to a wild-typestrain transformed with this plasmid (Fig. 2). However, thewild-type strain transformed with pNF2000 showed slightlydecreased UV resistance relative to that observed when itwas transformed with the cloning vector YEp24 (Fig. 2).This decreased UV resistance is not observed in the wild-type strain transformed with a plasmid containing a 4.5-kbpsubclone of the 9.7-kbp insert (see below) present in

0.0001 00 10 25 50

UV DOSE (J/m 2)FIG. 2. UV survival of yeast strains transformed with plasmids

YEp24 or pNF2000. Yeast strains transformed with the relevantplasmids were grown overnight in minimal medium to a density of 2X 107 to 4 x 107 cells per ml. Appropriate dilutions were then spreadonto agar plates containing minimal medium and exposed to UVradiation at the fluences indicated. Colonies were counted after 4days of incubation at 30°C. SX(YEp24), A; SX(pNF2000), A; rad2-2(YEp24), 0; rad2-2(pNF2000), 0; rad24(YEp24), O; rad2-4(pNF2000), M.

EcoRI

HindM

KpnI

3 EcoRI

XhoI

FIG. 3. Restriction map of pNF2000. Restriction enzyme siteswere mapped relative to known sites on the YEp24 vector. Theblack bar in the insert designates the location of the 4.5-kbpsubclone. The black bar in the vector indicates DNA from the yeast2p. circle.

pNF2000 (data not shown). The UV resistance of a rad24strain transformed with this plasmid was equivalent to thatof the wild-type strain containing the plasmid (data notshown).

Characterization of pNF2000 (i) Restriction analysis.Figure 3 shows a restriction map of plasmid pNF2000.Complete digestion with EcoRI enzyme yields four frag-ments with sizes determined by gel electrophoresis as 8.9,6.1, 2.1, and 0.35 kbp. From the additive size of thesefragments, we calculated that pNF2000 is 17.4 kbp in sizeand contains a DNA insert of 9.7 kbp.

(ii) Plasmid pNF2000 contains the RAD2 gene. Digestionwith HindIlI enzyme yields five fragments, the largest ofwhich is 9.3 kbp. The 9.3-kbp HindlIl fragment was clonedinto the vector YRp18 and shown to complement the UVsensitivity of rad2-2 and rad24. The fragment was trans-ferred into the integrating vector YIp5, and the resultingplasmid (pNF2012) was used to transform a rad2-2 ura3-52mutant. Eight stable URA+ transformants were isolated, sixof which were UV resistant, whereas two transformantsremained UV sensitive. To demonstrate that these stabletransformants contained integrated plasmid, DNA fromeach, as well as from rad2-2 and RAD+ nonintegrant strains,was restricted with the enzyme Sall. The restricted DNAwas electrophoresed on an agarose gel, transferred to nitro-cellulose, and hybridized to pBR322 DNA containing the9.3-kbp HindlIl fragment radiolabeled with 32P by nicktranslation. The integrating plasmid pNF2012 contains asingle Sall restriction site present in the vector. Hence, if theplasmid integrated at a unique site in the host genome(putatively the rad2-2 gene), we expected the labeled probeto hybridize to two fragments from the DNA digests of thestable transformants. Conversely, only a single hybridiza-tion band was expected with the RAD+ and rad2-2 noninte-grant DNA digests. Integration of the plasmid by homolo-gous recombination at the chromosomal URA3 gene isprecluded by the ura3-52 mutation in the strains used forintegration (12). This is confirmed in our own studies sincewe have failed to isolate stable integrants after transformingura3-52 strains with the plasmid YIp5 containing the URA3gene (data not shown). Figure 4 shows the results of typicalSouthern hybridization analysis and demonstrates that theadditive size of the two hybridization bands (25 kbp and 10.5kbp) from the DNA of transformants 1 and 2 (lanes 2 and 3)

0-

:D(/)

MOL. CELL. BIOL.

S. CEREVISIAE RAD2 GENE 293

A 1 2 3

28.0-23.7-

9.5-

FIG. 4. Southern hybridization analysis of integrant and controlstrains. Total genomic DNA was isolated from a wild-type strain andfrom two strains transformed with the integrating plasmid pNF2012.The DNA was restricted with Sail, and Southern hybridizationanalysis was performed as described in the text. X, molecular weightstandards from HindlIl restricted X DNA; lane 1, DNA from a wild-type strain; lanes 2 and 3, DNA from integrant strains.

differs from that of the single band from the nonintegrantDNA (21 kbp) by the size of the integrating plasmid (14.8kbp). An identical result was obtained with the two pheno-typically rad- stable transformants (data not shown). Wetherefore conclude that plasmid pNF2012 integrated into thegenome of all eight stable transformants and that the twoUV-sensitive integrants probably arose by gene conversionat RAD2. The observation that the Hindlll probe hybridizesto only a single fragment from the nonintegrant DNA digestsindicates that this 9.3-kbp fragment does not contain repeti-tive DNA sequences.

Having demonstrated that the integrating plasmid insertsinto a unique site in the yeast genome, we established bygenetic analysis that this site is at the RAD2 gene in the rad2-2 mutant. Three of the rad2-2 integrants were mated to aRAD+ strain, diploids were isolated and sporulated, and thespores were analyzed by conventional tetrad analysis. Table2 shows that of 25 complete tetrads examined from one ofthe crosses, the UV-resistance phenotype segregated 4+:0-in 24 tetrads, indicating a tight genetic linkage between theUV-resistance determinant on the cloned integrated DNAand the rad2-2 chromosomal gene. This result is expectedfrom integration by homologous recombination and demon-strates that the cloned DNA present in the integratingplasmid contains the yeast RAD2 gene. Unlinked markers inthis cross segregated 2+:2- (Table 2), and in a control crossbetween a rad2-2 nonintegrant strain and a RAD+ strain, theRAD2 marker also showed normal meiotic segregation (Ta-ble 2). Sporulation of the diploids obtained with the othertwo integrants yielded very poor viability, thus precludingtheir genetic analysis. However, tetrad analysis was carriedout with two other strains derived by integrating pNF2012into a rad24 mutant. After these integrants were mated to aRAD2 strain and the diploids were sporulated, the UV-resistance phenotype once again segregated 4 :0- in tetrads,whereas unlinked markers segregated 2+:2- (Table 2).

(iii) Localization of the RAD2 gene. In initial experiments,EcoRI fragments of 8.9 and 6.1 kbp from pNF2000 weresubcloned into the EcoRI site of a vector designated YRp17.These plasmids were shown to transform the rad2-2 ura3-52mutant to prototrophy for uracil but did not enhance the UVresistance of this strain, indicating that at least one of theclosely spaced EcoRI sites present in the cloned insert (Fig.3) lies within the RAD2 gene. To further localize the gene,plasmid pNF2000 was cut at the Sall and XhoI sites shown inFig. 3, and the complementary "sticky ends" were allowedto self-anneal. The resultant plasmid had a 3.1-kbp deletionof yeast insert DNA but still conferred normal levels of UVresistance to both the rad2-2 and rad24 mutants. Collective-ly, these observations localized the RAD2 gene to a 6.7-kbpfragment located between the Hindlll site and the XhoI site(Fig. 3). One end of the gene is situated between the EcoRIsite closer to the KpnI site and the XhoI site (Fig. 3).More definitive localization was achieved by subcloning

Sau3A partial digests of pNF2000. The 9.7-kbp clonedfragment contains no Sall sites (Fig. 3). We thereforeutilized a previously constructed vector (pNF2) specificallydesigned for the rapid subcloning and retrieval of DNAfragments devoid of Sall sites (7). A Sau3A partial digest ofthe 9.3-kbp HindlIl fragment was cloned into a uniqueBamHI site in pNF2, and plasmids were screened forcomplementation of rad2 mutants. The smallest subclonethus isolated was 4.5 kbp in size, and its location relative tothe original 9.7-kbp insert is shown in Fig. 3.

(iv) The RAD2 gene is not essential for viability. We havepreviously shown that the RAD3 gene of S. cerevisiae isessential for the viability of haploid cells in the absence ofDNA damage (8). We were thus prompted to determinewhether RAD2 is also an essential gene. A convenientmethod for establishing the essentiality of any yeast gene isto integrate an internal fragment, i.e., one missing both endsof the gene, into diploid cells that are homozygous for thegene in question (8, 9, 13). Integration by homologousrecombination of an internal fragment of the RAD2 geneshould inactivate one of the diploid chromosomal RAD2genes, and if the resulting mutation is lethal, sporulation ofthe diploid will generate only two viable haploid spores ineach tetrad (8, 9, 13).To carry out such an experiment, it was necessary to first

identify an internal fragment of the cloned RAD2 gene. Thedeletion analysis of pNF2000 described above located only

TABLE 2. Genetic analysis of integrant strainsMarker segregation

Cross Marker4-:0 3-:1 2-:2 1t:3 0:4-

rad2-2(pNF2012) x RAD+ RAD 24 1 0 0 0ADE 0 2 23 0 0MAT 0 2 23 0 0

rad2-2 x RADW RAD 0 1 13 0 0ADE 0 1 13 0 0MAT 0 1 13 0 0

rad24(pNF2012) x RAD+ RAD 6 0 0 0 0LYS 0 0 6 0 0

rad24(pNF2012) x RAD+ RAD 6 0 0 0 0LYS 0 0 6 0 0

rad24 x RAD+ RAD 0 0 11 0 0LYS 0 0 11 0 0

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294 NAUMOVSKI AND FRIEDBERG

Plasmid Fraqment Integrated

pNF2104

pNF 2101

KpnI EcoRII I I.I 1.5 2.3 4 1.3

Phenotype of Integrantrad- RAD+

Tetrad AnalysisSpore Viability Segregation of RAD in Complete Tetrads

40° 3:102 :20 1t 3° 0 400 7 6 3 2 0 0

7 6 6 5 1 0 0

20 22 7 1 1 0 0pNF2107

4:0Q 3:16 0

2:2- 1:3- 0:4-0 0 0

0 0 6 0 0

0 0 7 0 0FIG. 5. Analysis of diploid strains transformed with integrating plasmids containing fragments derived frori the 4.5-kbp subclone that

includes the RAD2 gene. The fragments are 1.0 (pNF2104) and 2.2 (pNF2101 and pNF2107) kbp in size. The diploids used for analysis of theRAD phenotype were heterozygous for RAD2(RAD21rad2-1). Those used for tetrad analysis were homozygous for RAD2(RAD21RAD2). Allrad haploid spores were shown to also be URA+. Experimental details are described in the text.

one end of the gene; hehce, such a fragment could not beconveniently recovered by restriction enzyme digestion atmapped sites. However, one can identify a RAD genefragment as being internal if, after its integration into theyeast genome, it converts a tJV-resistant RAD+Irad- het-erozygous diploid strain to UV sensitivity by inactivation ofthe wild-type gene in the diploid. Since integration can occurinto either the wild-type or mutant RAD2 sites with equalprobability, 50% of the integrants should be UV sensitive. ASau3A partial digest of the 4.5-kbp fragment (see above) wassubcloned into the integrating vector pNF3 (7), and threedifferent plasmids designated as pNF2101, pNF2107, andpNF2104, with inserts of 2.2, 2.2, and 1.0 kbp, respectively,were isolated (Fig. 5). When the former two plasmids wereindependently integrated into a RAD21rad2-1 heterozygousdiploid, an equal distribution of UV-resistant and UV-sensitive transfotmants was observed (Fig. 5), thus demon-strating that both 2.2-kbp fragments came from the interiorof the RAD2 gene. When either of these plasmids wasintegrated into a RAD21RAD2 homozygous diploid strain(this strain was used because it sporulates better than theheterozygous diploid), subsequent sporulation yielded fourviable spores in most tetrads, two of which were URA+ rad-(Fig. 5). The latter grew as well as the RAD+ spores isolatedfrom these tetrads. Furthermore, when the URA+ rad-haploids were mated to a rad24 haploid strain, the resultingdiploids were UV sensitive, indicating that the rad- pheno-type of the URA+ rad- haploids was due to inactivation ofthe RAD2 gene. These results demonstrate that the RAD2gene is not essential for the viability of haploid yeast cells.

Integration of plasmid pNF2104 containing the 1.0-kbpfragment into the RAD21rad2 heterozygous strain did notgenerate any UV-sensitive diploids (Fig. 5). In addition,sporulation of a RAD21RAD2 homozygous transformantyielded four RAD+ spores in each tetrad examined (Fig. 5).We therefore conclude that the 1.0-kbp fragment includesone end of the RAD2 gene.

(v) The RAD2 gene transcript. DNA-RNA hybridization bythe so-called Northern blotting technique revealed two RNAspecies that hybridized to a labeled DNA probe containingthe URA3 gene and an internal fragment of RAD2 (Fig. 6).One of these corresponds to the size of the yeast URA3 gene(0.9 to 1.0 kb) (M. Rose, Ph.D. thesis, MassachusettsInstitute of Technology; quoted in reference 4). The other is-3.2 kb in size and is presumed to be the RAD2 genetranscript. The intensity of the RAD2 hybridization banddetermined by autoradiography is less than that of the URA3band (Fig. 6), even though the size of the RAD2 fragment inthe labeled probe is more than twice that of URA3. Since thelatter gene is represented at the level of -5 to 10 transcripts

per cell in exponentially growing populations (M. Rose,Ph.D. thesis; quoted in reference 4), these observationssuggest that the RAD2 gene transcript is present at a lowerlevel. We noted that the RAD2 transcript runs just below thelarge ribosomal RNA. It is possible that the size andabundance of the transcript we measured may be distorted tosome extent by the presence of the excess ribosomal RNA.

(vi) Transformation of E. coli uvr- mutants with pNF2000.E. coli strains mutated in the uvrA, uvrB, or uvrC genes werenot complemented to UV resistance after transformationwith plasmid pNF2000 (data not shown); however, it was notindependently determined whether the RAD2 gene wasexpressed in these cells.

DISCUSSIONThe biochemistry of the incision of yeast DNA containing

bulky base damage such as pyrimidine dimers is apparentlycomplex, since at least five distinct genes (RADI, RAD2,RAD3, RAD4, and RADIO) are required for this process invivo (11, 16). None of the products of these genes have yetbeen isolated, nor has a damage-specific DNA incisingactivity been purified and characterized from any othereucaryotic source. To gain further insights into the biochem-istry of excision repair in eucaryotic cells, we isolated aseries of recombinant DNA plasmids that complement the

4.5-

RAD2-

2.2-

URA3-

FIG. 6. Northern analysis of the RAD2 transcript. Total RNAfrom strain S288C (RAD+) was subjected to DNA-RNA hybridiza-tion as described in the text. Molecular weight markers are fromDNA fragments (kilobases).

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S. CEREVISIAE RAD2 GENE 295

UV sensitivity of mutant strains defective in the RAD genesmentioned above. The present studies demonstrate that a9.7-kbp DNA insert present on a plasmid designatedpNF2000 includes the RAD2 gene. Genetic analysis showsthat an integrating plasmid containing a 9.3-kbp fragmentintegrates by homologous recombination exclusively at therad2 gene of mutant cells. Even though the integratingplasmid contains the yeast URA3 gene, integration at thelatter site is precluded because the rad2-2 and rad24 mu-tants both carry a mutation (ura3-52), which results ininsufficient homology with URA3 to permit integration at asignificant frequency (12).When three different allelic rad2 mutants are transformed

with pNF2000, the UV resistance of the mutants is restoredto the same level as that of wild-type cells transformed withthis plasmid. However, this level of resistance is slightlylower than that of wild-type cells transformed with just thecloning vector YEp24. This reduction in UV resistanceapparently results from cloned sequences outside of theRAD2 gene, since it is not observed when either rad2 orwild-type strains are transformed with a plasmid containing asmaller (4.5-kbp) fragment that includes RAD2. The natureof the flanking sequences presumably responsible for thiseffect has not been further investigated, nor has it beendetermined whether this effect is dependent on transforma-tion with multicopy plasmids.

Restriction mapping of pNF2000 revealed the absence ofSall sites in the 9.3-kbp HindIll fragment. This observationprovided the basis for a simple and rapid subcloning strategyin which Sau3A partial digests of this fragment were clonedinto a vector that contains a unique BamHI site flanked bySalI sites (7). DNA fragments cloned into the BamHI sitecan thus be conveniently excised by Sall digestion. Thesmallest subclone that completely complements the UVsensitivity of rad2 mutants contains a DNA insert of 4.5 kbp,which is consistent with the -3.2 kb size of the genetranscript estimated from DNA-RNA hybridization. Thissubclone should be convenient for further studies on theRAD2 gene, including DNA sequencing. Assuming that theRAD2 gene does not contain intervening sequences and thatthe coding region is about 3 kbp in size, then the molecularweight of the RAD2 gene product can be estimated to be-120,000.The RAD2 gene is not essential for the viability of haploid

cells. When one of the two RAD2 genes in a homozygousdiploid is inactivated by integration of an internal fragmentof the cloned gene, the haploid spores that result fromsporulation of the diploid have normal viability. This result isin contrast with that obtained from similar experiments withthe RAD3, actin, and tubulin genes, which have been shownto be essential genes in S. cerevisiae (8, 9, 13). The conver-sion of a UV-resistant (RAD+lrad-) heterozygous diploidstrain to UV sensitivity by inactivation of the single RAD+gene after integration of an internal gene fragment is asensitive and simple way of establishing that a cloned DNAfragment in an integrating vector is indeed derived from theinterior of the gene in question. The presence of either end ofthe gene in the fragment results in the regeneration of a wild-type gene following integration; hence, all diploid integrantsretain normal UV resistance. This general strategy should beuseful for identifying internal regions of any cloned yeastgene.

Estimates of the amount of RAD2 mRNA relative toURA3 mRNA present in unfractionated cell extracts suggestthat on the average there may be no more than 5 to 10 RAD2

transcripts per cell under normal growth conditions. We arecurrently investigating whether or not the RAD2 gene isinducible after DNA damage by UV radiation and by certainUV-mimetic chemicals. Studies are also aimed at placing thesubcloned gene under the control of a strong exogenousyeast promoter. Such gene tailoring should result in signifi-cant over-expression of the RAD2 gene in transformed yeastcells and will thus facilitate the isolation of adequate quanti-ties of RAD2 protein for biochemical investigations.

ACKNOWLEDGMENTS

We thank Glenn Pure, Eric Radany, Gordon Robinson, RogerSchultz, and Bill Weiss for helpful comments.

This research was supported by Public Health Service grant CA-12428 from the National Institutes of Health. L.N. is a trainee in theStanford University Medical Scientist Training Program, supportedby Public Health Service training grant GM-07365.

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VOL. 4, 1984