Proc. Nati. Acad. Sci. USAVol. 82, pp. 4763-4767, July 1985Genetics

Expression of a cloned denV gene of bacteriophage T4 inEscherichia coli

(T4 endonudease V/pyrimidine dimer DNA glycosylase/ultraviolet light/DNA excision repair/positive-selection vector)

KRISTOFFER VALERIE*t*, EARL E. HENDERSON*t, AND JON K. DE RIEL**Fels Research Institute and tDepartment of Microbiology and Immunology, Temple University School of Medicine, Philadelphia.PA 19140; and tDepartmentof Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44, Stockholm, Sweden

Communicated by Sidney Weinhouse, March 22, 1985

ABSTRACT A 713-base-pair Hae III fragment frombacteriophage T4 encompassing the denV gene with its preced-ing promoter has been cloned in a pBR322-derived positive-selection vector and introduced into a variety of DNA repair-deficient uvr and rec and uvr,rec Escherichia coli strains. ThedenV gene was found to be expressed, probably from its ownpromoter, causing pyrimidine dimer incision-deficient uvrA,uvrB, uvrC strains to be rescued by the denV gene. A uvrD(DNA helicase II) strain was also complemented, but to a lesserextent. A wild-type strain did not seem to be affected at the UVdoses tested. Surprisingly, all recA, recB, and recC strainstested also showed an increased UV resistance, perhaps byreinforcement of the intact uvr system in these strains.Complementation of denVW T4 strains and host-cell reactiva-tion of A phage was also observed in denV1 E. coil strains.Equilibrium sedimentation showed that DNA repair synthesisoccurred in a UV-irradiated uvrA E. coli strain carrying thecloned denV gene. Southern blotting confimed our earlierresults [Valerie, K., Henderson, E. E. & de Riel, J. K. (1984)Nucleic Acids Res. 12, 8085-8096] that the denV gene is locatedat 64 kilobases on the T4 map. Phage T2 (denV) did nothybridize to a denV-specific probe.

Endonuclease V, coded for by the denV gene of bacterio-phage T4 (1, 2), is a 16-kDa pyrimidine dimer-specific DNArepair enzyme with a pyrimidine dimer DNA glycosylaseactivity and an apurinic/apyrimidinic endonuclease activityin vitro (3-5). Only the DNA glycosylase has been shown toact in vivo (6), although the DNA sequence (7) and functionalproperties (4) of T4 denV amber strains support the conclu-sion that the denV gene product contains an apurinic/apyrimidinic endonuclease catalytic site. These two activitiesin conjunction make an incision 5' to a pyrimidine dimer inUV light-damaged DNA, initiating the removal ofthese lethallesions by the excision repair pathway induced in T4-infectedEscherichia coli (3, 4).Endonuclease V has been shown to complement

permeabilized E. coli cells with mutations in the excisionrepair genes uvrA, uvrB, and uvrC, and to some extent uvrD(DNA helicase II), indicating that the enzyme can replace theincision activity of the Uvr protein complex in E. coli (8).Remarkably, the T4 enzyme was able to restore the UVrepair capabilities of permeabilized (9, 10) or microinjected(11, 12) cells derived from humans with the autosomalrecessive inherited disease xeroderma pigmentosum. Suchexperiments have shown that all xeroderma pigmentosumcomplementation groups from A to G are complemented byT4 endonuclease V, as judged by restoration of unscheduledDNA synthesis (9, 12).

Since endonuclease V seems to be specific for pyrimidinedimers in UV-irradiated DNA (5) and acts on prokaryotic as

well as eukaryotic UV-damaged DNA in vivo, the protein isa promising tool for investigation of DNA repair in bothprokaryotes and eukaryotes.The cloning of the denV gene was reported by Lloyd and

Hanawalt (13), but their plasmids apparently were unstable invivo and were not thoroughly characterized. Their protocolfor isolating a cloned denV gene involved repeated selectionswith UV light, a potent mutagen that might induce and selectfor host gene revertants.As a step toward cloning the denV gene, we have previ-

ously published the mapping of the denV gene on the T4genome and its complete DNA sequence (7). Our mappingand DNA sequencing of the denV gene was recently con-firmed by Radany et al. (14). Their results were identical toours, except for a silent adenine to guanine difference in thethird position of the Val9 codon. In this report, we describethe successful cloning and expression of the denV gene in E.coli.

MATERIALS AND METHODSBacterial, Phage, and Plasmid Strains. A list of all bacterial,

phage, and plasmid strains used in this study is given in Table1. T2 DNA was purchased from Sigma.Media and Chemicals. Plasmid strains were usually grown

in LB broth (15) supplemented, when appropriate, withampicillin (50 ug/ml) and/or tetracycline (10 ,ug/ml). Allantibiotics and chemicals were purchased from Sigma orFisher, unless stated otherwise. Restriction endonucleaseswere obtained from New England Biolabs, Bethesda Re-search Laboratories, or Boehringer Mannheim and were usedas recommended by the manufacturers. T4 DNA ligase,bacterial alkaline phosphatase, and DNA polymerase I(Klenow) were purchased from New England Biolabs orBethesda Research Laboratories. DNA polymerase I(Komberg) came from Boehringer Mannheim. Cla I linkers(CATCGATG) were obtained from Bethesda Research Lab-oratories. [a-32P]dATP, [a-32P]dCTP (3000 Ci/mmol; 1 Ci =37 GBq) and [methyl-3H]thymidine (54 Ci/mmol) were pur-chased from Amersham. Kodak XAR-5 x-ray film was usedfor autoradiography with or without intensifying screens(DuPont).Growth and Purification of Deoxycytidine (dCyd) T4 Phage

and Plasmid DNA. The dCyd T4 quadruple mutant strain wasgrown in E. coli K803 and E. coli B834 as described by Snyderet al. (16), except that LB broth was used instead of M9Smedium. Cleared lysates ofplasmid-containing E. coli strainswere produced essentially as described (15).

Transformation ofE. coil. Transformation ofE. coli strainswas performed as described (17), except that the cells wereshocked at 370C instead of at 420C.

Abbreviations: bp, base pair(s); kb, kilobase(s); dCyd, deoxycyti-dine.

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Table 1. Bacterial, phage, and plasmid strains

Strain Relevant genotype Source

E. coliAB1157 uvr+,rec+ E. A. Adelberg*AB2480 uvrA6,recA13 P. Howard-Flanders*AB2463 recA13 P. Howard-Flanders*AB2470 recB2l P. Howard-Flanders*AB3058 recC22 P. Howard-Flanders*JC5547 recA13,recB21,recC22 A. J. Clark*JC5519 recB21,recC22 A. J. Clark*AB1886 uvrA6 P. Howard-Flanders*AB1885 uvrBS P. Howard-Flanders*AB1884 uvrC34 P. Howard-Flanders*SK728 uvrD3,uvr253 S. KushnerAB2500 uvrA6,thyA15 P. Howard-Flanders*CR63 supD R. Appleyard*K803 r17,m,,rg1-,su+ R. MarshB834 r;,m-,galUrgl+,su R. MarshMM294 rk ,m + M. Meselson*

T4 phageT4 56(amE51)denA(nd28) R. Marsh

denB(ArIIH23B)alc8T4v1 denV- W. HarmfarP13 denV- (del) D. H. Hall

X phageW30 cI M. Uhldn

PlasmidspBR322 amp+,tet+ Laboratory stockpUN121 amp+,tet(ind) B. NilssonpdenV-7 amp+,tet+,uv+ This study

pUN121::713bp T4 Hae IIIfragment

pdenV-52 amp' pBR322::447bp T4 Hinfl This studyfragment (Cla I linkers)

*Obtained from B. Bachmann (Genetic Stock Center, YaleUniversity).

Detection of T4 DNA in Plasmid Clones. After transforma-tion of E. coli strains with recombinant plasmids, colonieswere transferred to nitrocellulose filters (Schleicher &Schuell) and processed for hybridization as described (18).For restriction enzyme analysis of purified DNA samples,digested DNA was subjected to agarose gel electrophoresis,then blotted onto nitrocellulose filters and hybridized todenatured 32P-labeled probe as described (19).UV Irradiation. Phage and bacteria were irradiated at 254

nm by using a fixed UV lamp (Model UVGL-58), and theincident dose rate was measured by a Black-Ray photometer(both from Ultraviolet-Products, San Gabriel, CA). Bacteriawere irradiated directly on agar plates after dilution andspreading. Phage were irradiated (-2 X 108 plaque-formingunits per ml) in SM buffer (15) on a Petri dish, and aliquotswere taken at various times before adding bacteria and softagar. Care was taken not to expose bacteria or phage tovisible light after UV irradiation, to prevent photoreactiva-tion.Measurement of Repair by Equilibrium Sedimentation.

Measurement of repair synthesis was performed essentiallyaccording to Pettijohn and Hanawalt (20) with slight modifi-cations (21). Briefly, overnight cultures of AB2500/pUN121(denV-) and AB2500/pdenV-7 (denV+) were grown in M9Smedium (15) supplemented with 0.2% glucose/thiamine (1,ug/ml)/thymidine (100 ttg/ml)/ampicillin (50 ug/ml) (com-plete medium). The cells were harvested, washed, andresuspended at 1 x 108 cells per ml in complete medium,

except that 5-bromodeoxyuridine (BrdUrd) at 10 ,g/ml wasadded in place of thymidine. The cells were incubated for 15min prior to irradiation with UV light (254 nm) for a total doseof 80 J/m2. [methyi)H]Thymidine (54 Ci/mmol, 10 ,Ci/ml)was immediately added and the cells were incubated at 37°Cfor 90 min, then washed and lysed with lysozyme andNaDodSO4. The lysate was treated with RNase A, proteinaseK, and thereafter was phenol extracted. The DNA wassubjected to equilibrium sedimentation by centrifugation in aBeckman SW50.1 rotor at 30,000 rpm for 60 hr at 25°C.

RESULTSCloning of the denV Gene. We have sequenced a large

portion of the T4 genome from the denV gene region ofbacteriophage T4 (7). The restriction enzyme Hae III cuts oneach side of the denV gene within adjacent genes, whilepreserving the denV gene with its putative promoter in a713-base-pair (bp) fragment. A partially purified fractioft ofHae Ill-digested dCyd T4 DNA containing fragments of -700bp was eluted from a 4% polyacrylamide gel and blunt-endligated into equimolar amounts of phosphatase-treated SmaI-digested pUN121. The vector pUN121 (22) is a positiveselection vector carrying a tetracycline gene under thecontrol ofthe bacteriophage X cI gene. The Sma I cloning siteis in the cI structural gene. The ligation mixture was trans-formed into E. coli MM294 and selected on tetracycline-containing LB plates. Clones that were resistant to bothampicillin and tetracycline, indicating that they contained aninsert in the cI genie, were screened for the denVgene by filterhybridization to nick-translated 1.9-kilobase (kb) T4 EcoRIfragment [62.1-64.0 kb on the T4 map (23)] containing the 3'portion of the denV gene. The plasmid DNA from fourpositive clones was extracted and transformed into E. coliAB2480 (uvrA, recA) to assay for enhanced UV resistance.Of the four clones that were positive for the denV gene insert,one (pdenV-7) conferred enhanced UV resistance on E. coliAB2480 after UV irradiation. The map of pdenV-7 shown inFig. 1 was obtained from restriction enzyme analysis andSouthern blots (Fig. 2). To make a denV-specific probe,plasmid pdenV-52 was constructed from the 713-bp HaeIIIfragment by digesting with Hinfl, which will cut between thefirst and second codons of the denV gene and 33 bp down-stream from the gene, creating a 447-bp fragment (7). By the

FIG. 1. Plasmid map of pdenV-7 (denV+). Map shows the 713-bpHae III fragment (shaded) inserted in the Sma I site ofpUN121 (22).Parentheses indicate that the sites are lost. The direction oftranscrip-tion of the denV gene is indicated with an arrow.

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A 1 2 3 4 5 6 7 8 9

23kb- ioomb9.4 ~ am

6.7 v4.4 _ _,

2.3 -

1.6 -

101112 13

0.9

0.5

- a0.2

BSallXholXbalPstl

15.215.1 ..0 .

14.8I 1.5 ~.

45 55 60G--OM 2R

75kb

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map 63 64 6Skb

Genetic denV WI iilimap p

cHailllHinfl

713 transcription

"7

FIG. 2. Southern blot of dCyd T4, T2, and pdenV-7 DNA. (A)DNA (5 ,ug) isolated from dCyd T4 (denV), T2 (den V-), as well asfrom pdenV-7 (denV+) (1 Ag) was digested with various restrictionenzymes and fractionated on a 0.85% agarose gel. The DNA wasblotted onto a nitrocellulose filter and hybridized to 32P-labeled T4DNA probe derived from the insert of pdenV-52, specific for thedenV gene. DNA marker consisted of a mixture of HindIII-digestedX phage, BstNI-digested pBR322, and Hinfl-digested pBR322. Lanes1-8 contain dCyd T4 DNA, lane 9 contains T2, and lanes 10-13contain pdenV-7 DNA. Lanes: 1, Sal I; 2, Xho I; 3, Xba I; 4, Pst I;5, Sal I/Xho I; 6, Xba I/Xho I; 7, Pst I/Xho I; 8-10, Taq I; 11,BamHI/Xho I; 12, HindI11; 13, Xho I. (B) The published physicalmap of the denV region of phage T4 (23, 24). Enlarged Taq Irestriction map is obtained from sequencing studies (refs. 7 and 14,and unpublished data). Thicker lines indicate fragments observed inA that hybridize to the denV probe. The fragment size (kb) is alsoshown for each fragment. The genetic map of the denV gene (7) andthe ipIl and ipIII genes, which have both been sequenced (unpub-lished results) are indicated together with their respective promoters(P) and the orientation of transcription. (C) Cloned fragmentscontaining the denV gene. The 713-bp Hae III fragment contains thedenVpromoter and structural gene. The 447-bpHinfl fragment contains

only the denV structural gene and was cloned in the Cla I site ofpBR322after addition of Cla I linkers to the filled-in Hinfl ends.

addition of Cla I linkers to the filled in Hinfl sites, theinitiation codon was restored. The fragment was ligated intothe Cla I site of pBR322, resulting in a convenient cartridgeconsisting of the denV structural gene.

Survival of Various UV Light-Sensitive E. coli StrainsCarrying a Cloned denV Gene. To assess whether the en-hanced UV irradiation resistance observed for E. coliAB2480/pdenV-7 was due to a plasmid-encoded gene, weextracted the plasmid from this UV light-resistant clone andtransformed it into a variety of DNA repair-deficient E. colistrains listed in Table 1. As controls, we used the parentvector pUN121, and a pdenV-7 derivative, pdenV-7(Xho),which carries an inactivated denV gene. To generate pdenV-

7(Xho), pdenV-7 was digested with the restriction enzymeXho I, which cuts in the middle of the denV gene (7) (Fig. 1),and the ends were filled in with DNA polymerase I (Klenow)and re-ligated. This procedure will create a +4 frameshiftmutation in the denVgene, thereby inactivating the gene. Thenegative controls, pUN121 and pdenV-7(Xho) in E. coliAB2480, had virtually identical rates of survival after expo-sure to UV light, as can be seen in Fig. 3A. E. coli AB2480containing no plasmid was found to have the same survivalrate as AB2480/pUN121 and AB2480/pdenV-7(Xho).We found that pdenV-7 enhanced the survival of every

UV-sensitive E. coli strain tested. E. coli AB2480 (uvrA,recA) showed the greatest difference of -3 orders of mag-nitude at a UV dose of 1 J/m2 when compared to a controlcontaining pUN121 (Fig. 3A). E. coli strains AB1886 (uvrA),AB1885 (uvrB), and AB1884 (uvrC) all showed about thesame enhancement when carrying a cloned copy of the denVgene (Fig. 3B). SK728(uvrD)/pdenV-7 was somewhat moresensitive to UV irradiation than the corresponding uvrA,uvrB, and uvrC/pdenV-7 clones, while the controlSK728(uvrD)/pUN121 was somewhat more resistant to UVlight than uvrA, uvrB, and uvrC controls (Fig. 3B). E. coliAB2500 (uvrA, thyA)/pdenV-7 also acquired resistance toUV light, while AB2500/pUN121 retained the expectedsensitivity to UV light (Fig. 3B). E. coli strains having recA,recB, recC, and combinations of these mutations were alsopartially rescued by pdenV-7 (Fig. 3 A and C).Complementation of T4 denV- Strains by a Cloned denV

Gene. If pdenV-7 contains the denV gene, an E. coli straincarrying this plasmid ought to complement T4 denV- strainswhen plated on such a strain. To test this, we transformed E.coli CR63 (supD) with pdenV-7 and pUN121, respectively,and then infected with either UV-irradiated dCyd T4(denV+), T4,1 (denV-), or farP13 (denV-) phage. Both theT4v, and the farP13 strains were partially rescued by thecloned denV gene (Fig. 4). At 12 J/m2, the cloned geneincreased T4v, survival =5-fold andfarP13 survival -25-fold.The cloned denV gene did not seem to have any effect on thewild-type dCyd T4 strain at the UV doses tested.

Host-Cell Reactivation of A Phage inE. coli Strains Carryinga Cloned denV Gene. A UV repair-deficient E. coli host mustbe used to assess the effect of a cloned denVgene on host-cellreactivation of X phage, since the host DNA excision repairsystem is not inactivated during X phage infection (25). TheuvrA, recA, as well as the uvrA repair-deficient E. coli hoststested showed an increased host-cell reactivation of X phagewhen they carried a cloned denV gene (Fig. 5).Measurement of Repair by Equilibrium Sedimentation. To

show that the enhanced survival following UV irradiation ofE.coli strains containing a cloned denV gene was due to excisionrepair, E. coli AB2500 (uvrA, thyA) carrying pdenV-7 orpUN121 were assayed for excision repair after UV irradiation.Without irradiation, both strains incorporated Brd-Urd-[3H]dThd extensively as indicated by the peak of radioac-tivity at an increased density (Fig. 6 A and B). As expected,AB2500/pUN121 incorporated hardly any counts after UVirradiation (Fig. 6D), presumably because ofthe lack ofincisionat pyrimidine dimers in this strain. After UV irradiation, E. coliAB2500/pdenV-7 incorporated BrdUrd-[3H]dThd in its paren-tal DNA, as indicated by the lighter DNA peak (Fig. 6C),showing restoration of repair synthesis in this strain.Southern Blot of Phage and Plasmid DNA. To show that

pdenV-7 carries an insert from the correct region on the T4genome, a Cla I fragment derived from pdenV-52 (Table 1 andFig. 2C) specific for the denV gene was used as a hybridizationprobe for a Southern blot (19) of dCyd T4 DNA digested withvarious restriction enzymes, all making cuts at well-characte-rized sites along the T4 genome (23, 24). We also includeddigested pdenV-7 and T2 DNA. The blot in Fig. 2A clearlyshows that the denV gene is located at 64 kb on the T4 map,

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FIG. 3. Survival following UV irradiation of DNA repair-deficient E. coli strains containing a cloned copy of the denV gene. DNArepair-deficient E. coli strains (Table 1) were transformed withpdenV-7(denVA)(solid symbols), and pUN121 (denV-), [open symbols, excepto, which indicates AB2480/pdenV-7(Xho) (denV-)]. Saturated overnight cultures were serially diluted in SM buffer, plated on ampicillin agarplates and irradiated with the indicated doses of UV light (254 nm). Plates were incubated at 37°C in the dark overnight and the colonies werecounted the next day. Survival curves were constructed from several independent experiments and from several different cell dilutions. Averagevalues were taken for each point. None of the clones resistant to UV light was subjected to UV irradiation prior to the experiments. (A) E. colistrain AB2480 (uvrA, recA) (o, *), AB2463 (recA) (A, A), JC5547 (recA, B. C) (o, *), and AB2480 containing pdenV-7(Xho) denV- (o). (B) E.coli strain AB1886 (uvrA) (o, *), AB1885 (uvrB) (o, *), AB1884 (uvrC) (,*,SK728 (uvrD) (v, v), and AB2500 (uvrA, thyA) (o, *). (C) E.coli strain AB2470 (recB) (o, *), AB3058 (recC) (o, *), JC5519 (recB, recC) (, A), and AB1157 (wild type) (v, v).

because the probe hybridizes to the characteristic completeand partial restriction fragments predicted from Fig. 2B. Theblot in Fig. 2A also shows the hybridization to the 282- and283-bp Taq I doublet from dCyd T4 DNA spanning the denVgene (7, 14). The Taq I doublet is also found in pdenV-7, inaddition to the expected 262-bp HindIII and 1424-bp XhoI/BamHI fragments (Fig. 1). In the case ofDNA from phageT2, which does not seem to have any denV gene (26), the

100'

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4 8

UV dose, J/m212

FIG. 4. Plaque-forming ability of denV- T4 strains after UVirradiation. E. coli CR63/pUN121 (denV-; open symbols), and E.coli CR63/pden-7 (denV+; solid symbols), were infected with UV-irradiated T4,1 (o, 0), farP13 (o, *) (both denV-), and dCyd T4(denV+; A, A) and incubated at 37°C overnight. The next day, thenumber of plaques was counted and survival curves were plotted byusing the average number from several experiments and dilutions.

probe hybridizes only very weakly to a Taq I fragment of -2kb.

DISCUSSIONThe data we present here show that we have succeeded incloning the denV gene in E. coli. Clones were screened

100

10

0 10 20 30UV dose, J/m2

FIG. 5. Host-cell reactivation of phage X in E. coli strainscarrying a cloned denV gene. Phage X W30 (cI) was irradiated in SMbuffer in a Petri dish, with the indicated UV doses (254 nm). Atdifferent intervals, aliquots were removed and added to E. coli cells.Open symbols indicate E. coli strains carrying no plasmid [except A,which indicates AB2480/pUN121 (denV-)], and solid symbols indi-cate E. coli strains transformed with pdenV-7 (denV+). AB1157(uvr+, rec+) (v), AB1886 (uvrA) (o, *), AB2463 (recA) (o), andAB2480 (uvrA, recA) (o, e).

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FIG. 6. Measurement of repair by equilibrium sedimentation. E.cli AB2500/pdenV-7 (denV+) (A and C) and AB2500/pUN121(denV-) (B and D) were grown in BrdUrd, UV irradiated with a totaldose of 80 J/m2 (C and D), or left unirradiated (A and B) before[3H]dThd was added to assay repair synthesis. o, Absorbance at 260nm (1 OD26 unit = 50 Ag/ml);*, precipitable cpm. Fractions werecollected from the bottom of the tubes.

without UV selection, thereby avoiding the possibility of UVlight-induced reversion of the host organism. The gene isexpressed, probably from its own promoter, and confersresistance to UV light upon UV repair-deficient E. colistrains, as we have been able to demonstrate by all DNArepair criteria tested. Resistance to UV irradiation iscotransferred with the antibiotic resistance marker of theplasmid pdenV-7, containing the denV gene. This plasmid isstably maintained in all E. coli strains we have tested anddoes not show any of the deletions or rearrangementsreported for other cloned T4 fragments by others (13) and byus (7). At this point, we cannot say whether the choice ofcloning vector has made any difference in the stability of thedenV gene. Our own preliminary attempts to redone thedenV gene in other plasmid vectors suggest that under certainconditions the denV gene is unstable, perhaps becauseendonuclease V interferes with some E. coli function(s). Ifthat is the case, pdenV-7 may be stable because the orien-tation of the denV gene in this construction is opposite to theorientation of the cI gene, into which the denV gene is cloned(Fig. 1). This could be the reason why only one of four clonesthat were transformed into E. coli AB2480 (uvrA, recA)conferred resistance to UV light.As expected, the denV gene rescued uvrA, uvrB, uvrC, and

uvrD mutants of E. coli in our study. This is consistent withthe effect of endonuclease V on permeabilized DNA repair-deficient E. coli strains (8) and with the results of infection ofT4 phage inactivated by UV irradiation into repair-deficientE. coli strains (27). Surprisingly, the denV gene also rescuedrec- strains, in contrast to previous observations (8, 27). Themost likely explanation of our results is that the denV genesimply reinforces the intact uvr system in rec- cells. TheRecA protein, and possibly also the RecB, RecC complex(exonuclease V), are required for the induction of the uvrgenes during the UV light-induced SOS response in E. coli(28). Supplementation by the denV gene product of thenoninducible host excision repair pathway in rec- cells couldthus account for the increased resistance to UV light.

Neither colony-forming ability nor host-cell reactivationwas restored to wild-type levels in our study. The most likelyexplanation is the inability of endonuclease V to specifically

recognize UV light-induced lesions other than pyrimidinedirers.The cloned denV gene also partially rescued the UV-

irradiated denW T4 strainsT4v, andfarPJ3, demonstratingcomplementation of denVWT4 phage by the plasmid-carrieddenV gene. It is not clear why complementation does notreach the wild-type level of survival in these experiments. Aplausible reason might be that the infecting T4 phage degradeE. coli chromosomal and plasmid DNA within minutes,because of the T4 denA gene product (29), thereby reducingthe efficiency of complementation by the plasmid-carrieddenV gene.The availability of the cloned denV gene should facilitate

assessment of such questions as the importance of theapurinic/apyrimidinic endonuclease activity of endonucleaseV in vivo and the effect of denV gene expression on E. ccliDNA metabolism in general. It may also be possible todevelop expression vectors allowing overproduction of endo-nuclease V for physical and enzymatic studies, to search forrelated genes in other organisms, and to explore the effect ofdenV gene expression in eukaryotic cells.

We thank B. Nilsson and M.Uhlen for plasmid and phage strains,B. Bachmann and S. Kushner for providing E. ccli strains, and GregHarvey for preparation of the manuscript. This work was supportedby a Basic Research Grant to J.K.deR. from the March of DimesBirth Defects Foundation. These data were in part presented at theNinth International Congress on Photobiology, July 1-6, 1984,Philadelphia, PA.

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