JOURNAL OF BACTERIOLOGY, Nov. 1987, p. 4884-48920021-9193/87/114884-09$02.00/0Copyright © 1987, American Society for Microbiology

Vol. 169, No. 11

Mutational Inactivation of the Saccharomyces cerevisiae RAD4Gene in Escherichia coli

REINHARD FLEER,t WOLFRAM SIEDE, AND ERROL C. FRIEDBERG*

Department ofPathology, Stanford University School of Medicine, Stanford, California 94305

Received 22 April 1987/Accepted 7 August 1987

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotideexcision repair. When plasmids containing the wild-type gene were transformed into various Escherichia colistrains, transformation frequencies were drastically reduced. Most plasmids recovered from transformantsshowed deletions or rearrangements. A minority of plasmids recovered from E. coli HB101 showed no evidenceof deletion or rearrangement, but when they were transformed into S. cerevisiae on centromeric vectors, littleor no complementation of the UV sensitivity of rad4 mutants was observed. Deliberate insertional mutagenesisof the wild-type RAD4 allele before transformation ofE. coli restored transformation to normal levels. Plasmidsrecovered from these transformants contained an inactive rad4 allele; however, removal of the inserted DNAfragment restored normal RAD4 function. These experiments suggest that expression of the RAD4 gene is lethalto E. coli and show that lethality can be prevented by inactivation of the gene before transformation.Stationary-phase cultures of some strains of E. coli transformed with plasmids containing an inactivated RAD4gene showed a pronounced delay in the resumption of exponential growth, suggesting that the mutant (and, byinference, possibly wild-type) Rad4 protein interferes with normal growth control in E. coli. The rad4-2,rad4-3, and rad44 chromosomal alleles were leaky relative to a rad4 disruption mutant. In addition,overexpression of plasmid-borne mutant rad4 alleles resulted in partial complementation of rad4 strains. Theseobservations suggest that the Rad4 protein is relatively insensitive to mutational inactivation.

The RAD4 gene of Saccharomyces cerevisiae is one of fivegenes required for the incision of damaged DNA duringnucleotide excision repair in S. cerevisiae (5, 6, 22, 27). Themolecular cloning and detailed characterization of this genehave been complicated by the inability to identify recombi-nant plasmids which complement the UV sensitivity of rad4mutants after the propagation of yeast genomic libraries inEscherichia coli, (4, 21, 24). Recent studies in this laboratoryhave demonstrated that the RAD4 gene is situated immedi-ately upstream of the SPT2 gene and that an inactive rad4allele is contained on integrating and centromeric plasmidsthat include STP2 (4, 23). A functional RAD4 gene can berestored by creating defined gaps in a mutant allele carriedon a centromeric plasmid vector and by repairing the gaps invivo by transferring information from wild-type genomicyeast sequences (4, 19). However, complementing activity islost if such gap-repaired plasmids isolated directly fromyeast cells are passaged through E. coli HB101 beforetransformation of rad4 mutants. On the basis of these andother results, we concluded that plasmids containing RAD4cannot be passaged in E. coli HB101 without sufferingmutational inactivation of the gene (4).

In the present study, we investigated the fate of gap-repaired plasmids in various E. coli strains, including arecBC sbcB mutant known to tolerate eucaryotic sequencesthat are unstable in standard E. coli hosts (2, 15, 28). Weshow that in all strains tested, the efficiency of transforma-tion with plasmids containing a functional RAD4 gene ismarkedly reduced relative to that of plasmids with aninactivated gene and that the majority of plasmids isolatedafter transformation of E. coli contain deletions and rear-rangements. Plasmids with no detectable deletions or rear-

* Corresponding author.t Present address: Genetica, 94340 Joinville-le-Pont, Paris

France.

rangements are nonetheless inactivated, presumably bypoint mutations or small deletions. However, insertion ofextraneous DNA fragments into selected unique restrictionsites in the cloned wild-type RAD4 gene isolated directlyfrom yeast cells inactivates the gene and permits its ampli-fication in E. coli. After amplification, the inserted fragmentscan be removed to restore a functional RAD4 gene whichfully complements the UV sensitivity of rad4 mutants.The inability to propagate RAD4-containing plasmids in E.

coli apparently reflects a toxic effect of Rad4 protein ratherthan perturbation of a specific sequence in the RAD4 gene.In a finding consistent with this interpretation, we show inthe present study that several mutant rad4 alleles whichretain partial complementing activity in yeast cells alsointerfere with normal growth of E. coli.

MATERIALS AND METHODSStrains and growth conditions. The yeast and E. coli strains

used in this study are listed in Table 1. The growth condi-tions have been described previously (4).Measurement of growth kinetics of plasmid-transformed E.

coli strains. Cultures of various E. coli strains were trans-formed with plasmids YCp53, pNF422, or pNF422GR-INBgland grown overnight in LB medium (1% tryptone [DifcoLaboratories], 1% sodium chloride, 0.5% yeast extract[Difco]) in the presence of tetracycline (12.5 jig/ml). Nosignificant differences in cell number were detected in sta-tionary-phase cultures. The cultures were diluted 1:250 inLB medium and incubated in the presence (and in somecases, in the absence) of tetracycline at 37°C with constantagitation. Samples were removed at frequent intervals, andthe growth of the cultures was monitored by measuring theoptical density at 600 nm.Measurement of UV survival. Semiquantitative survival

tests were carried out as previously described (4). Forquantitative analyses, single yeast colonies were picked

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RAD4 GENE OF S. CEREVISIAE 4885

TABLE 1. Yeast and E. coli strainsStrain Relevant genotype Source

YeastSX46a a RAD his3-532 ade2 ura3-52 trpl-289 Our laboratory stocksrad4-2 a rad4-2 his3-532 ade2 ura3-52 trpl-289 G. Pure, this laboratoryrad4-3 a rad4-3 his3-532 ade2 ura3-52 trp1-289 G. Pure, this laboratoryWS8104-2B a rad44 ade2-1 ura3-52 trpl-289 W. Siede, Gesellschaft fur Strahlenforschung,

Neuherberg, Federal Republic of Germanyrad4-O0a a rad4-10 his3-532 ade2 trpl-289 R. Fleer, this study

E. coliHB101 recA hsdR hsdM Our laboratory stocksRR1 recA+ hsdR hsdM Department of Genetics, Stanford University,

Stanford, Calif.TG1 recA+ hsdR Our laboratory stocksCES201 A(srl-recA)306::TnJO (Tetr) recB21 recC22 sbcB15 hsdR F. Stahl, University of Oregon, PortlandDB1161 recA56 srl::TnJO (Tetr) recB21 recC22 sbcB15 hsdR hsdM D. Botstein, Massachusetts Institute of

Technology, CambridgeVC257 hsd-53 Statagene Cloning SystemsMM294 endAl hsdR M. Rosenberg, Smith Kline Beckman Corp.JM103 endAl sbcB15 hsdR4 Our laboratory stocks

a RAD4 disruption derivative of strain SX46a.

from YPD (1% yeast extract, 2% peptone, 2% dextrose)plates, suspended in distilled water, and spread on YPDplates for exposure to UV radiation, as previously described(4). The presence of plasmids in transformed cells wasevaluated before UV irradiation by replica plating on selec-tive medium. This parameter was also monitored afterirradiation so that the quantitative analysis of UV sensitivitycould be corrected for plasmid loss. Replica plating underselective conditions also facilitated detection of spontaneousstable integrants in colonies transformed with YRp-typeplasmids. Integrant colonies are faster growing and formunsectored colonies.

Purification of plasmid DNA and other recombinant DNAprocedures. Small- and large-scale preparations of plasmidDNA from E. coli (1, 11, 14), isolation and purification ofplasmid DNA from S. cerevisiae (4), and purification ofrestriction fragments by electroelution from agarose gels (4)were carried out as described previously. E. coli was trans-formed by the standard calcium chloride procedure (3).Yeast cells were transformed by the method of Ito et al. (12)or the spheroplasting technique of Hinnen et al. (10). Gaprepair of linearized plasmnids in yeasts was carried out aspreviously described (4, 19). Restriction enzymes werepurchased from Bethesda Research Laboratories or NewEngland BioLabs, Inc. T4 DNA ligase and E. coli DNApolymerase I (Klenow fragment) were obtained fromBethesda Research Laboratories, and calf intestinalphosphatase was obtained from Boehringer MannheimBiochemicals. All enzymes were used according to theinstructions of the manufacturers.Plasmid constructions. (i) Construction of the multicopy

plashiid pNF422GR carrying a wild-type RAD4 allele. Thederivations of the various plasmids used in this study arediagrammed in Fig. 1. Plasmid pNF422 is a multicopyYRp-type plasmid containing an inactivated rad4 allele. Toconstruct this plasmid, the centromeric vector YCp5O (13)was deleted of its single Bglll and PvuI sites to generateplasmid YCp53. This deletion was achieved by cutting thesesites with the appropriate restriction enzymes, filling in thesticky ends, and ligating the blunt-ended molecules (4). A3.3-kilobase (kb) PstI-BamHI fragment from plasmidpNF411GR-EC1 (4), which contains an inactivated allele ofRAD4, was cloned into the SmaI site of YCp53 to generatepNF417 (Fig. 1A). A derivative of plasmid pNF417

(pNF421; Fig. 1A) carries TRPI instead of URA3 as aselectable marker. This plasmid was generated by cloning a1.2-kb Stul-BamHI fragment containing the TRPI gene fromYRp7 into a blunt-ended unique NcoI site in pNF417. Thisprocedure regenerated the NcoI site at the NcoI-BamHIboundary. The removal of a XbaI-XhoI fragment containingCEN4 from pNF421 deleted the centromere and generatedthe multicopy plasmid pNF422 (Fig. 1A). In some experi-ments, plasmid pNF422 was converted back to the centro-meric form by reinserting CEN4 (contained on an HpaI-XmnI fragment) from YCp5O into the NcoI site in pNF422.This centromeric derivative of pNF422 is designatedpNF422CEN (Fig. 1A). A restriction map of a derivative ofplasmid pNF422 (see below) is shown in Fig. 2 and includessites relevant to the cloning steps described above.The site of inactivation of the RAD4 gene in an ancestor of

the multicopy plasmid pNF422 was previously localized to a100-base-pair (bp) region contained on an XmnI-PvuI frag-ment (4). Deletion of a 350-bp PvuI-BglIl fragment immedi-ately flanking this region, followed by in vivo gap repair in anappropriate yeast strain, was shown to restore a wild-typeRAD4 allele at a high frequency (4). To carry out gap repairof the mutated RAD4 allele carried on plasmid pNF422, thelinearized plasmid deleted of this 350-bp region wastransfected into rad4-10 (a UV-sensitive mutant in which thechromosomal RAD4 gene was disrupted by insertion of theURA3 gene at a unique MstII site [4] [Fig. 2]).The recovery of the gap-repaired plasmid (designated

pNF422GR; Fig. 1A and B) from Trp+ yeast transformantswas greatly facilitated by the high copy number. However,confirmation of the successful repair of the BglII-PvuI gapwas complicated by the fact that the mutant rad4 allele inpNF422 complements rad4 mutants to nearly wild-typelevels when it is present in multiple copies (see Results).Therefore, pNF422GR transformants of strain rad4-2 werescreened for cells in which the plasmid was integrated. Suchintegrants (containing a single copy ofpNF422GR) exhibiteda UV resistance phenotype, indicating that the multicopyplasmid pNF422GR contains a wild-type RAD4 allele. Ad-ditionally, a centromeric derivative of the multicopy plasmidpNF422GR was independently derived in two experimentsand was shown to fully complement the UV sensitivity ofrad4 mutants (see Results).

(ii) Construction of plasmids containing insertional muta-

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pNF411 GR-EC a rad4

YRp7 - . TRP1

YCp53

pNF417

-F1pNF421

CEN4 4-

pNF422 CEN pNF422

pNF422 GR

pNF422 GR

a 11 1frag.

pNF422 GR-IN BgI

CEN4 10 BoLi II frag.

I p

pNF422 CEN-GR-IN Bgl * pNF422 CEN-GR1

Y.UIrag.

pNF422 CEN-GR-IN Bgl/ IN Pvu

frag. Edfrag.

pNF422 CEN-GR-IN Pw pNF422 CEN-GR2

FIG. 1. Schematic representation of the various plasmids used and their derivations. GR, Gap repair; CEN, centromeric single-copyvector; INBgl, insertion of extraneous DNA in the RAD4 BglIl site; INpU, insertion of extraneous DNA in the RAD4 PvuI site; frag.,fragment. See text for details.

tions in the RAD4 gene. To generate a reversible insertionalmutation in the wild-type RAD4 gene, a 400-bp BglII frag-ment from the unrelated yeast RAD2 gene was cloned intothe unique BglII site in plasmid pNF422GR (Fig. 2). Thisfragment is out of frame with the RAD4 translational codeand contains multiple stop codons. E. coli HB101 wastransfected with a ligation mixture containing plasmidpNF422GR cut at the BglII site and the 400-bp BglII linearfragment. The plasmid recovered from E. coli was desig-nated pNF422GR-INBgl (Fig. 1B). Plasmid pNF422GR-INBglwas converted to the centromeric form by the insertion ofCEN4 to yield pNF422CEN-GR-INBgl (Fig. 1B and 2).Removal of the 400-bp BglII fragment from this plasmidgenerated pNF422CEN-GR1, containing the wild-typeRAD4 gene (Fig. 1B).A second insertional mutation in plasmid pNF422CEN-

GR-INBgl was created by cloning a 1.8-kb PvuI fragmentfrom plasmid pUC119 into a unique PvuI site (Fig. 2), whichgenerated the centromeric plasmid pNF422CEN-GR-

INBgl/INpvu (Fig. 1B and 2). Removal of the BglII insertyielded the centromeric plasmid pNF422CEN-GR-INpvu,carrying an insertional mutation at the PvuI site, and re-moval of the PvuI fragment yielded pNF422CEN-GR2 (Fig.1B). This plasmid was expected to be identical to plasmidpNF422CEN-GR1 (see above).

RESULTS

Transformation of E. coli with plasmids containing thewild-type RAD4 gene. In initial experiments, a centromericplasmid (designated pNF402GR for [gap repair]) containing awild-type RAD4 allele was generated by gap repair in yeastcells (4). We isolated this plasmid directly from yeast cells,as described previously (4), and transformed rad4-2, rad4-3,and rad44 mutant strains both before and after propagatingthe plasmid in E. coli HB101. In a finding consistent with theresults of previous studies (4), when this plasmid was notpropagated in E. coli HB101, all Ura+ transformants tested

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FIG. 2. Restriction map of plasmid pNF422CEN-GR-INBgj/INpNu, a derivative of plasmid pNF422GR. The arrow indicates thetranscriptional orientation of the RAD4 gene. The BglII and PvuI fragments used for insertional mutagenesis of pNF422GR are shown. TheAmpr gene is shown in brackets because the gene was inactivated by removing a PvuI site (also shown in brackets). The CEN sequence wascloned into the plasmid after insertional mutagenesis of RAD4 to facilitate propagation in E. coli.

were UV resistant (data not shown). However, none of therad4 mUtants transformed with plasmids isolated from E.coli were UV resistant.The majority of the clonal isolates from E. coli contained

plasmids with deletions of as much as 75% of the originalplasmid (Table 2, classes II to IX). In most cases, theplasmid restriction patterns were also indicative of extensiverearrangements (Table 2, classes II to IX). These deletionsand rearrangements were not strictly random, since somerestriction fragments are common to several classes ofplasmids (Table 2, classes II to VI). Additionally, eightindependently isolated transformants of E. coli HB101 con-tained centromeric plasmids without detectable deletions orrearrangements (Table 2, class I). These plasmids can bestably propagated and are collectively designatedpNF402GR-EC plasmids. We previously demonstrated thatthe pNF402GR-EC class of plasmids does not complementthe UV sensitivity of any rad4 mutants examined (4).

Similar experiments were carried out with multicopyplasmids carrying RAD4. Plasmid pNF422 (Fig. 1A) is amulticopy derivative of a particular class I plasmid(pNF411GR-EC [Fig. 1A]) originally isolated from E. coli

HB101 (4). Plasmid pNF422 was also gap repaired in vivo.The resulting plasmid, isolated directly from yeast cells, wasdesignated pNF422GR (Fig. 1). Comparison of the restric-tion pattern of plasmid pNF422GR (which contains a wild-type RAD4 allele) with the restriction pattern of pNF422(which contains an inactivated rad4 allele) did not revealdetectable deletions or rearrangements (Fig. 3). However, aswas the case with the centromeric gap-repaired plasmidpNF402GR (see above), transformation of E. coli HB101with the multicopy gap-repaired plasmid pNF422GR wasassociated with extensive deletion and rearrangement (datanot shown).To determine whether inactivation of the RAD4 gene in E.

coli is strain specific, we introduced the centromeric plasmidpNF402GR (which contains the wild-type RAD4 gene) intotwo recA recB recC sbcB hsdR mutants (CES201 andDB1161) (Table 2) and into the recA+ recBC+ strain TG1(data not shown). In all cases, the transformants yieldeddeleted or rearranged plasmids. Inactivated, nonrearrangedplasmids were not isolated from these strains (Table 2).However, fewer of these transformants were examined thanin the case of E. coli HB101; hence, this result may simply

TABLE 2. Deletion and rearrangement of plasmid pNF402GR after propagation in various E. coli strainsa

No. of bacterial clones *Siz of AmountNo.ofbactacPlasmid PstI restriction fragments (kb) Sze ° of DNArecovered in strains: class plasmid deleted

HB101 CES201 DB1161 0.9 1.2 1.5 1.6 1.8 2.3 2.4 3.0 3.2 3.6 3.8 4.2 4.3 4.8 5.8 6.8 (kb) (kb)

8 0 0 ib + + + + 14.0 NDC0 2 1 II + + + + 11.0 3.01 0 0 III + + + 10.6 3.42 0 0 IV + + + 8.0 6.00 3 0 V + + + 7.7 6.30 1 0 VI + + + 4.8 9.2

13 0 2 VII + 4.8 9.21 0 0 VIII + 4.3 9.71 0 0 IX + 3.6 11.4

Results shown are from eight independent transformations carried out with two different preparations of purified plasmid pNF402GR isolated from a UV-resistant rad44 strain after gap repair in vivo. See text for details.

b Plasmids were subjected to additional extensive restriction enzyme digestion and were indistinguishable from plasmid pNF402GR.c ND, Not detected.

r

0IL-x -'a -M-PI_ (a

=_ -E CCcc a> El0L.-.(

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7.1 -6.1 -5.1 -4.1 -

3.1 -

2.0-

1.6 -

M 1 2 1 M

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FIG. 3. Restriction digests of the multicopy plasmid pNF422before and after gap repair in yeast cells. pNF422 is a derivative ofpNF402GR-EC1 (4) and contains the same mutant rad4 allele. Therestriction fragments shown are the products of digestion with BglIIand BamHI, both of which cut pNF422 once but leave the 2,umDNA intact. M, Molecular weight markers. Lane 1, PlasmidpNF422 propagated in E. coli HB101; lane 2, plasmid pNF422GRisolated directly from yeast cells after gap repair in vivo. Theadditional bands in lane 2 are the 2,Lm DNA. The major band of the2pum DNA is indicated by the arrow.

reflect a sampling bias. Transformation of the recBC strainDB1161 with plasmid pNF402GR-EC1 (which contains amutant rad4 allele) resulted in frequent deletions, althoughthis plasmid can be stably propagated in E. coli HB101.Hence, there are strain differences with respect to thetolerance of plasmids even when they contain inactivatedrad4 alleles. The phenomenon of inactivation is apparentlyspecific to plasmids containing the RAD4 gene, since unre-lated control plasmids similar in size to plasmids of thepNF402GR-EC class were stably propagated in all strainsexamined.

In addition to various deletions and rearrangements ofplasmid DNA, we consistently observed that the transfor-mation of E. coli HB101, CES201, DB1161, and TG1 witheither centromeric or multicopy plasmids containing a wild-type RAD4 gene occurred at a frequency 1O-3 to 10-4 lowerthan that with plasmids containing an inactivated rad4 allele.

Propagation of plasmids in E. coli by reversible inactivationof the RAD4 gene. The inability to amplify plasmids contain-ing a wild-type RAD4 gene places considerable restraints onthe characterization of the cloned gene, particularly thedetermination of its nucleotide sequence. To overcome thisproblem, we explored a strategy of deliberate mutationalinactivation of the wild-type gene isolated directly fromyeast cells by inserting an extraneous DNA fragment into aselected unique restriction site, with the expectation that thiswould preclude additional inactivating events during thepropagation of plasmids in E. coli. Removal of the insertedfragment from amplified plasmid DNA should then restorethe wild-type RAD4 gene.A centromeric gap-repaired plasmid was constructed with

a 400-bp DNA insert at a unique BglII site (Fig. 2). Thisplasmid was propagated in E. coli HB101 and was designatedpNF422CEN-GR-INBgl (Fig. 1B). As anticipated, this plas-mid did not complement the UV sensitivity of any mutantrad4 strains tested (Fig. 4). However, when the plasmid wascut with BglII to excise the 400-bp insert and then recircular-ized by ligation in vitro, full complementation of rad4mutants was observed (Fig. 4).To demonstrate the generality of this strategy, we con-

structed a second rad4 insertional mutation by using a 1.8-kbfragment ofDNA cloned into a different site in the gene. For

these experiments, we used the previously inactivatedcentromeric plasmid PNF422CEN-GR-INBgl. The 1.8-kbDNA fragment was inserted into a unique PvuI site in therad4 gene (Fig. 2), and the plasmid was propagated in E. coli.After the isolation of plasmid DNA, the 400-bp BglII insertwas removed and the religated plasmid was again propagatedin E. coli. This plasmid (designated pNF422CEN-GR-INpvU;Fig. 1B) also failed to complement the UV sensitivity of therad4-2 mutant (Fig. 4). However, after digestion with PvuIand religation in vitro, rad4 transformants with wild-typelevels of UV resistance were recovered (Fig. 4). Thesetransformants showed strict cosegregation of the TRPI andRAD4 plasmid markers, indicating that UV resistance wasdue to expression of a plasmid-borne determinant (data notshown).Leakiness of rad4 alleles. Transformation with the

multicopy plasmid pNF422 resulted in a marked increase inUV resistance of rad4-2 (Fig. 5), rad4-3, and rad4-4 (data notshown) strains that approached wild-type levels. Thiscomplementation was specific to rad4 mutants; no enhancedUV resistance was observed after the transformation ofother mutants from the RAD3 epistasis group (data notshown). These experiments indicate that the product of therad4 mutant allele in plasmid pNF422 retains residual activ-ity when the product is overexpressed from a multicopyplasmid. This result is particularly surprising because it hassince been determined that the RAD4 allele in plasmid

100O

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C',

0 20 40 60

UV Dose (J/m 2FIG. 4. Quantitative survival of UV-irradiated strains trans-

formed with various plasmids. Symbols: ED, Rad+ strain SX46a; A,strain rad4-2 transformed with plasmid pNF422CEN-GR1; O, strainrad4-2 transformed with plasmid pNF422CEN-GR2; +, Rad-rad4-2 strain; A, rad4-2 strain transformed with plasmidpNF422CEN-GR-INBgi; *, rad4-2 strain transformed with plasmidpNF422CEN-GR-INp>U. See text for details.

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1001

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UV Dose (J/m 2)FIG. 5. Quantitative survival of UV-irradiated strains trans-

formed with various plasmids. Symbols: EB, Rad+ strain SX46a; +,Rad- strain rad4-2; A, rad4-2 strain transformed with plasmidpNF422; *, rad4-2 strain transformed with plasmid pNF422CEN;x, Rad- disruption mutant rad4-10; *, rad4-10 strain transformedwith plasmid pNF422CEN.

pNF422 carries a frameshift mutation that is expected toencode a truncated polypeptide about half the size of thenative Rad4 protein (L. Couto and E. C. Friedberg, unpub-lished data).The partial complementation of rad4 mutants by plasmid

pNF422 raised the question whether the gap-repaired plas-mid pNF422GR indeed contained a wild-type RAD4 allele(see Materials and Methods). This was independently deter-mined by isolating rad4 mutant strains that contain a singleintegrated copy of plasmid pNF422GR and demonstratingthat these integrant strains are fully UV resistant (data notshown). Additionally, as noted above, centromeric deriva-tives of pNF422 propagated in E. coli as insertional mutantsfully complement the UV sensitivity of rad4 mutants afterthe inserted fragments are removed.

Close inspection of the chromosomal rad4 mutant allelesdesignated rad4-2, rad4-3, and rad44 indicates that they tooare leaky. We previously generated a number of insertionmutants containing URA3 disruptions of the chromosomalgene (4). In the present study, we examined the phenotype ofone of these strains (rad4-10) and observed that this mutantis considerably more sensitive to killing by UV radiationthan the rad4-2 (Fig. 5), rad4-3, and rad44 (data not shown)point mutants. Indeed, the UV sensitivity of this disruptionmutant is comparable to that of mutants with deletions inother RAD genes required for the incision of DNA duringnucleotide excision repair in S. cerevisiae (7, 9, 18, 20, 25,29).

Using the highly UV-sensitive rad4-10 mutant, we showedthat the rad4 mutation in plasmid pNF422 is leaky evenwhen the plasmid copy number is reduced. When acentromeric derivative of pNF422 (pNF422CEN; Fig. 1A)was transformed into rad4-10, partial complementation ofUV sensitivity to the level of a rad4-2 mutant was clearlyevident (Fig. 5). A low level of residual complementingactivity was also observed after rad4-10 was transformedwith the centromeric plasmid pNF422CEN-GR-INBgl (datanot shown). Since the rad4 allele in this plasmid carries aninsertional mutation, the gene product of this mutant is alsoexpected to be a truncated polypeptide.Growth kinetics of E. coli strains transformed with plasmids

carrying mutant rad4 alleles. In view of the observationthat the mutated rad4 alleles in plasmids pNF422 andpNF422CEN-GR-INBgl (expected to encode truncatedpolypeptides) retain partial activity in S. cerevisiae, weexplored the possibility that these mutated rad4 alleles areinformative indicators of the mechanism whereby the wild-type allele inactivates E. coli. We therefore transformedvarious E. coli strains with the multicopy plasmid pNF422 orpNF422GR-INBgl and examined the kinetics of the resump-tion of exponential growth in stationary-phase cultures di-luted into fresh medium. In E. coli HB101 and the isogenicrecA+ derivative strain RR1, plasmid pNF422GR-INBgl hadno detectable effect on growth relative to the plasmid vectorYCp53 (Fig. 6A and B). Cells transformed with plasmidpNF422 showed a very slight delay in the resumption ofexponential growth kinetics. All the other E. coli strainsexamined (Fig. 6C to F), particularly E. coli MM294 (Fig.6E), showed a significant delay in the resumption of expo-nential growth after transformation with this plasmid. Oncelogarithmic growth resumed, however, the kinetics were notsignificantly different from that of control cells. The growthinhibition caused by plasmid pNF422 was not the result ofplasmid loss and consequent sensitivity to tetracycline, sincesimilar growth kinetics were observed in the absence of theantibiotic (Fig. 6C). A less-pronounced growth delay wasalso observed in some (but not all) strains transformed withplasmid pNF422GR-INBgl (Fig. 6C to E).

DISCUSSION

RAD4 is one of at least five genes absolutely required forexcision repair of bulky DNA adducts in the yeast S.cerevisiae (5, 6, 21, 27). Isolation of the RADI, RAD2,RAD3, and RADIO genes was readily accomplished bycomplementing appropriate mutants with plasmids from ayeast genomic library amplified in E. coli (7-9, 17, 18, 20, 25,29, 30). Extensive screening of such libraries failed to yieldplasmids which complement the three rad4 mutants tested(4, 21, 24). On the other hand, screening a yeast genomiclibrary not previously propagated in E. coli yielded a plasmidwhich fully complements such mutants (24). When centro-meric plasmids containing a wild-type RAD4 gene are trans-formed into E. coli HB101, plasmids recovered with appro-priately sized recombinant inserts contain inactivated rad4alleles. A wild-type allele can be restored by gap repair inyeast. However, transformation into E. coli again results ininactivation of the RAD4 gene (4). All of these observationsare consistent with the conclusion that plasmids containing awild-type RAD4 gene cannot be propagated in E. coli with-out inactivation of the gene.The present study shows that plasmids containing a wild-

type RAD4 allele are not tolerated in any of the E. coli

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hat 37 °CFIG. 6. Growth kinetics of logarithmically growing cultures of different E. coli strains transformed with plasmid YCp53 (O), pNF422 (* ),

or pNF422GR-INBgl (U). Transformed cultures were grown overnight to stationary phase and then diluted 1:250 in fresh medium withtetracycline (or, in the experiment shown in panel C [A], without tetracycline). (A) Strain RR1; (B) strain HB101; (C) strain TG1; (D) strainVCS257; (E) strain MM294; (F) strain JM103. OD 600 nm, Optical density at 600 nm.

strains examined. The overall yield of transformants isdrastically reduced (by a factor of 103 to 104), and mostplasmids that are recovered have extensive deletions orrearrangements. A minority of the plasmids recovered fromE. coli HB101 show no detectable alteration by restrictionanalysis, and when they are present in low copy numbers(i.e., as centromeric plasmids), they do not effect significantcomplementation of the UV sensitivity of several relativelyleaky rad4 mutants. This observation suggests that in aminority of plasmids, inactivation of the RAD4 gene in E.coli HB101 results from (presumably random) point muta-tions and is consistent with the absence of alterationsdetectable by restriction analysis of DNA.

One of these mutant alleles has been investigated moreextensively and apparently encodes a truncated polypeptidewith residual complementing activity that can be readilydetected when phenotypic complementation is examined in ahighly UV-sensitive rad4 mutant. Indeed, when it is ex-pressed from a multicopy plasmid, this mutant form of Rad4protein complements chromosomal rad4 mutants close towild-type levels. This leakiness notwithstanding, in view ofthe poor transforming efficiency of E. coli by plasmids witha wild-type RAD4 allele, it is unlikely that a yeast genomiclibrary serially propagated in E. coli would be well repre-sented with such mutant plasmids. This provides a reason-able explanation of the failure to detect complementation of

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RAD4 GENE OF S. CEREVISIAE 4891

rad4 mutants with multicopy plasmid libraries propagated inE. coli HB101.

Plasmids can be stably propagated in E. coli HB101 whenthey contain a rad4 allele inactivated by deliberate inser-tional mutagenesis. Under these conditions, there is appar-ently no further inactivation of the gene in E. coli, sincereversal of the insertional mutation restores normal RAD4activity. Reversible insertional mutagenesis can be effectedat two different sites in the coding region of the geneseparated by 350 bp, which suggests that mutagenic inacti-vation of the wild-type RAD4 gene in E. coli is not the resultof degradation of a specific sequence in the gene (e.g., ahairpin loop). This inference in consistent with the observa-tion that inactivation of the wild-type gene is not mitigatedby the propagation of plasmids in strains defective in recB,recC, and sbcB genes. Such mutant strains are permissivefor the propagation of other plasmids containing eucaryoticsequences which cannot be propagated in standard E. colihost strains (2, 15, 28).

Collectively, these results are consistent with the hypoth-esis that intolerance of the RAD4 gene in E. coli is the resultof Rad4 protein toxicity. At present, the molecular basis forthis toxicity is not known. However, the present studydemonstrates that even mutant forms of the protein interferewith normal growth of E. coli cells. Specifically, we notedthat when stationary-phase cells transformed with plasmidsbearing mutant rad4 alleles were diluted into fresh medium,there was a strain-dependent delay in the resumption oflogarithmic growth. However, once exponential growth re-sumed, the kinetics were essentially similar to those ofcontrol cells. This observation suggests that the productsencoded by the mutant rad4 genes interefere with metabolicevents critical for the initiation of exponential growth instationary-phase cultures. In E. coli strains transformed withmutant rad4 alleles, this interference is apparently tempo-rary, which posssibly reflects the fact that the mutant proteinis eventually displaced or neutralized. This model predictsthat in cells transformed with plasmids carrying the wild-type RAD4 gene, the effect of the protein encoded by thisallele is much more severe and results in a permanentinhibition of growth in transformed cultures. This interpre-tation is consistent with the noted strain dependency forgrowth inhibition, since one would expect that differentstrains of E. coli might neutralize the toxicity of mutant Rad4protein with various efficiencies. The strain dependence ofgrowth inhibition also provides a reasonable explanation forthe observation that a plasmid which contains an inactivatedRAD4 allele, and hence is tolerated in E. coli HB101, isnonetheless deleted and rearranged when it is transformedinto E. coli DB1161.

In E. coli MM294 transformed with plasmid pNF422, thekinetics of resumption of exponential growth are particularlyslow. This characteristic provides a simple and quick screenfor chromosomal mutants that are resistant to the toxic effectof the mutant rad4 allele carried on this plasmid. In prelim-inary experiments, such a screen has yielded several faster-growing variants which are currently under detailed investi-gation. The availability of mutants totally resistant to thetoxic effect of the Rad4 protein would be of obvious utilityfor propagating RAD4-containing plasmids and additionallymight provide more substantial insights into the mechanismof this toxicity.The present study highlights earlier observations that the

rad4-2, rad4-3, and rad44 point mutants are considerablyless UV sensitive than RADI, RAD2, or RAD3 mutants orRAD4 or RADIO disruption or deletion mutants (7, 9, 16, 20,

25, 29). The rad4-3 mutation can be partially suppressed byknown ochre suppressor tRNA genes and is likely an ochremutation (21). Since normal tRNAGIA can function as aweak ochre suppressor in S. cerevisiae (21), it is possiblethat leakiness of the rad4-3 allele reflects partial suppressionby the multiple copies of the tRNAGIA gene known to bepresent in the yeast genome (21) or by other suppressionmechanisms (26). The rad4-2 and rad44 alleles are notsuppressed by ochre suppressors (21). However, we cannoteliminate the possibility that they too are subject to alterna-tive forms of partial suppression in yeast cells. These con-siderations notwithstanding, the relative UV resistance of allthree rad4 mutants is in striking contrast to that of othermutant genes in the RAD3 epistasis group. Extensive muta-genesis of the RAD3 gene has shown that many missense andall nonsense mutations completely inactivate excision repairactivity in yeast cells (16). Similarly, most radl and rad2mutants are highly UV sensitive (7, 9, 18, 29). A number ofplasmid-borne mutant rad4 alleles also encode proteins withresidual activity, especially when overexpressed. This ob-servation suggests that the Rad4 protein is relatively insen-sitive to conformational changes resulting from mutationalalteration. These properties are not those expected of aprotein with catalytic activity and suggest that the Rad4protein may have a different role during the incision of DNAcontaining bulky base damage in yeast cells.

ACKNOWLEDGMENTS

We gratefully acknowledge advice and criticism from LouieNaumovski, Bill Weiss, Linda Couto, Helmut Burtscher, JaneCooper, David Kalainov, Clare Lambert, Paul Saxon, and RogerSchultz. We also thank Jean Oberlindacher for typing the manu-script.These studies were supported by Public Health Service research

grant CA 12428. R.F. was supported by a postdoctoral fellowshipfrom the Deutscher Akademischer Austauschdienst, and W.S. wassupported by a postdoctoral fellowship from the North AtlanticTreaty Organization given by the Deutscher AkademischerAustauschdienst.

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