Vol. 168, No. 2JOURNAL OF BACTERIOLOGY, Nov. 1986, P. 1014-10180021-9193/86/111014-05$02.00/0Copyright © 1986, American Society for Microbiology

Expression of the Bacteriophage T4 denV Structural Gene inEscherichia colit

ADRIAN RECINOS III,1 MARY LOU AUGUSTINE,2 KAY M. HIGGINS,2 AND R. STEPHEN LLOYD2*

Departments of Microbiology1 and Biochemistry, Center in Molecular Toxicology, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232

Received 13 May 1986/Accepted 25 August 1986

The expression of the T4 denV gene, which previously had been cloned in plasmid constructs downstream ofthe bacteriophage A hybrid promoter-operator oLpR was analyzed under a variety of growth parameters.Expression of the denV gene product, endonuclease V, was confirmed in DNA repair-deficient Escherichia coli(uvrA recA) by Western blot analyses and by enhancements of resistance to UV irradiation.

The bacteriophage T4 genome contains a series of DNArepair genes (denV, uvsW, uvsX, and uvs Y), the products ofwhich catalyze the removal of DNA damage introduced bychemicals and irradiation (5). The product of the denV gene,endonuclease V, is an enzyme which functions in an excisionDNA repair pathway specific for removal of UV-introducedpyrimidine dimer photoproducts (6, 11-14, 16, 20). Lloydand Hanawalt (10) first reported the cloning and expressionof the denV gene in a uvrA recA Escherichia coli strain.These transformants showed enhanced resistance to UVirradiation and also conferred increased plaque-forming abil-ity to both UV-irradiated phage A and a T4 mutant defectivefor denV. The plasmid constructions which contained thedenV gene, however, were not stably maintained withinthese cells.The sequencing of overlapping DNA fragments encom-

passing several kilobases of the T4 genome in the region ofthe denV gene has resulted in identification of the codingsequence of the gene (15, 18). Within the last year, stableexpression of the denV gene from a plasmid construct inDNA repair-deficient E. coli was reported (4, 19). Theresults concerning UV survival, phage X host cell reactiva-tion, and denV complementation were remarkably similar tothose reported by Lloyd and Hanawalt (10).

Efforts in our laboratory to clone the complete denV genein multicopy plasmids by inserting segments of the T4genome spanning the gene were not successful. However,the 3' portion of denV was cloned as a gene fusion with lacZand detected as a P-galactosidase-endonuclease V fusionprotein with antibodies directed against endonuclease V (9).Recently we have used the technique of oligonucleotidesite-directed mutagenesis to create new DNA restrictionsites closely flanking the endonuclease V coding sequence(1Sa). This strategy resulted in stabilization of the denVstructural gene within a multicopy expression vector(pGX2608, a gift of Genex Corp.). The denV insert wasplaced behind phage X regulatory sequences in the correctreading orientation (pGX2608-16-denV+ [Fig. 1, inset]) andin the reverse orientation (pGX2608-19-denV) (1Sa). Hereinwe report on the expression of the denV gene product fromthis plasmid construct in a variety of E. coli genotypicbackgrounds and culture conditions.

* Corresponding author.t This report is dedicated to the memory of William E. Recinos,

M.D. (1949-1985).

The initial E. coli strain into which pGX2608-16-denV+and pGX2608-19-denV were inserted was GX1200, which isa defective A lysogen carrying the c1857 gene which encodesa temperature-sensitive repressor. Since the denV gene waspositioned downstream of the A leftward operator and right-ward promoter, expression of endonuclease V should becontrolled by shifts in growth temperature. When 30°C brothcultures (LB medium supplemented with 100 ,ug of ampicillinper ml) of these transformants in exponential growth wereshifted to 42°C for 15 min before a 2-h expression period at37°C, intracellular accumulation of endonuclease V was notdetected by Western blot analysis (3, 8, 9, 17). Control lanescontaining a partially purified preparation of endonuclease Vfrom T4-infected cells were positive in these experiments.Subsequent studies concerning the effects of differentgrowth temperatures on the expression of endonuclease V inother E. coli strains (see below) provided an explanation forthis lack of product accumulation.To examine both the expression of endonuclease V in a

nonrepressed E. coli host and the effects of denV clones oncell survival after UV irradiation, the denV+ and denVplasmids were each transformed into E. coli AB2480 (uvrArecA) and AB2487 (uvr+ recA). Although these recipientstrains do not carry a cI gene, transformation frequenciesand restriction enzyme analyses of plasmids indicated thatunrepressed expression from OLPR was neither lethal totransformants nor deleterious to plasmid stability.UV dose-response data indicated that the presence of the

denV+ plasmid in E. coli defective for both excision repairand recombination (strain AB2480) enhanced survival bymore than 4 orders of magnitude relative to the parentalstrain when cells in stationary-phase growth at 30°C weresubjected to irradiations of 0.15 J/m2 (Fig. 1, squares).Similar but less exaggerated survival increments were alsodemonstrated when the denV+ plasmid was carried by themore UV-resistant E. coli AB2487 (Fig. 1, circles). No suchenhancements were seen for AB2480 or AB2487 cells whichharbored the denV plasmid. Parallel experiments with cellswhich were irradiated during exponential growth producedvery similar dose-response profiles, although the UV doserequired to kill any given fraction of repair-competent cellsappeared, surprisingly, to be slightly greater than that re-quired for the same cells in stationary phase (data notshown).The T4v1 mutant, which is unable to synthesize wild-type

endonuclease V, is much more sensitive to inactivation byUV irradiation than is the T4D wild-type phage. To further

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NOTES 1015

1.0 CIaIEcoRi EcoRI

E

.001Cl-

pGX26O8-1~~~~~Amp"deno01

0

.01=

.001=

I

.1 .3 ,5 .7 .9

UV,J/m2

FIG. 1. Colony-forming ability of UV-irradiated DNA repair-deficient E. coli containing denV+ and denV plasmid constructs.Stationary-phase cells were irradiated on agar plates and incubatedat 30°C for 36 h. Symbols: O, AB2480 (uvrA recA)(pGX2608-19[denVJ); E, AB2480(pGX2608-16 [denV+]); 0, AB2487 (recA)(pGX2608-19 [denV]); 0, AB2487(pGX2608-16 [denV+]). Inset:schematic diagram of plasmid pGX2608-16-denV+.

assess the production of endonuclease V in E. coli harboringthe denV+ plasmid, tests were conducted to determinewhether uvr recA denV+ E. coli could rescue the excisionrepair defect in UV-irradiated T4v,. Mutant and wild-typephage were UV irradiated at various doses and used in 30°Cinfections of E. coli AB2480 which incorporated the denV+plasmid, the denV plasmid, or merely pGX2608 (containingno denV insert). Exponential-phase cells were grown in LBmedium (100 ,ug of ampicillin per ml) at 30°C. Analyses ofthese data showed that the mutant phage was fully comple-mented to wild-type levels of UV resistance (Fig. 2, closedcircles). No complementation was observed in infections ofcontrol strains unable to express the denV gene product.

Since significant enhancements of UV resistance wereafforded by introduction of the denV+ plasmid in repair-deficient E. coli strains, Western blot analyses were con-ducted to determine whether immunoreactive endonucleaseV could be detected within these cells. Transformants bear-ing the denV+ plasmid were grown to stationary phase atvarious temperatures and in different media, and intracellu-lar proteins were subjected to Western blot analysis. Themajor findings of these experiments are illustrated in Fig. 3.The peak in accumulation of endonuclease V appeared onWestern blots for cultures grown at 25°C (Fig. 3, 25°C, lane3) and, surprisingly, it disappeared in cases in which 0.5%glucose was added to cultures of these cells (lanes 4 and 6).Since effects of catabolite repression were not expected for

this O[PR expression system, experiments were conducted tofurther define the phenomenon by examining the influence ofincreasing glucose concentrations on both intracellular ac-cumulation of endonuclease V and survival after UV irradi-ation. Concentrations of glucose of 20.05% markedly re-duced the visualization of endonuclease V on Western blots(Fig. 4, inset), whereas glucose at 0.05 and 0.1% had noeffect on UV survival profiles. When cell survival wasexamined for these cells in the presence of a more substan-tial glucose addition (0.4%), UV resistance decreased appre-ciably in the supplemented culture (Fig. 4). Additionally,glucose concentrations which inhibited detection of endonu-clease V on Western blots similarly inhibited detection ofanother protein band (at about 40 kilodaltons) which isroutinely visualized when crude anti-endonuclease V anti-serum preparations are used in these procedures. (Thesesera contain numerous preimmune rabbit anti-E. coli anti-bodies.) Dramatic effects on the detection of endonuclease Vby Western analysis were not seen when growth media weresimilarly supplemented with glycerol (lanes 9 to 17).These observations prompted a survey of host strains

intended to identify those which lead to increased accumu-lation of endonuclease V. The denV+ plasmid was trans-

100

10

1.0- A

0.1

3.0 6.0 9.0 12.0UV, J/m2

FIG. 2. Complementation of UV-irradiated T4 denV phage.Wild-type T4 phage (open symbols) and T4v1 phage (closed sym-bols) were UV irradiated at various doses and used to infect thefollowing E. coli strains: 0 and *, AB2480(pGX2608-16 [denV']); Aand A, AB2480(pGX2608-19 [denV]); O and *, AB2480(pGX2608).

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232 34 5 620°*C-

43 O- _

25.7 _

18.4-- _1F4,3-

4 5 6300C

43.0-

25.7-

18.4-

14.3-

1 2 3 4 5 6 1 2 3 4 5 6

25CI2 3 4 5 6250C 135

43.0-

25.7-

18.4-

14.3-

1 2 3 4 5 6 C 2 3 4 S 6*w} 7r:,,..-t I

1 2 3 4 5 6

* ;,,F- s e:w.;: a,-

..f:

43.0-

25.7-

18.4-

14.3-

FIG. 3. Effects of temperature and growth media on accumulations of endonuclease V within E. coli AB2480 harboring pGX2608-16(denV+). Cells were grown to stationary phase at 20, 25, 30, and 35°C, and proteins were processed for silver stain and Western blotanalyses. Lanes: 1, partially purified preparation of endonuclease V from T4-infected E. coli; 2, AB2480(pGX2608-19 [denV]) (negativecontrol grown in LB medium at 30°C for all gels); 3 to 6, AB2480(pGX2608-16 [denV+]) grown in LB, LB-0.5% glucose, 2x yeasttryptone, and 2x yeast tryptone-0.5% glucose, respectively. The protein positions and molecular weights (in thousands) of the standardsare indicated.

formed into a variety of E. coli strains which were known tobe deficient in various proteolytic pathways (Table 1,GW5837-5865, B). Western blot analyses revealed that noneof the E. coli strains tested accumulated endonuclease V atlevels greater than that found for the repair-defectiveAB2480 strain. Another endeavor to increase the expressionof endonuclease V in E. coli involved the use of oligonucle-otide site-directed mutagenesis to alter two AUA isoleucinecodons to the AUC triplet. For highly expressed E. coligenes, the latter Ile codon is utilized substantially morepreferentially than is the AUA triplet (7). Western blotanalyses measuring accumulations of endonuclease V ob-tained with the Ile-altered constructs, however, demon-strated endonuclease V bands of intensities similar to thoseseen for cells containing the nonmutagenized denV+ plas-mid.

Collectively, these data demonstrated that under optimalgrowth conditions T4 endonuclease V accumulated to levelsgreater than that re'quired for maximal enhancement of UVsurvival. Observations supporting this assertion are as fol-lows. (i) When UV survival was analyzed for cells whichboth harbored the denV+' plasmid and accumulated immu-nologically detectable levels of endonuclease V, the dose-response profiles had initial shoulders which transited tostraight-line inac-tivation curves with increasing UV doses.The usual interpretation for this "shoulder repair" is thatinitially introduced DNA lesions are subject to efficient

repair but the repair system becomes saturated with furtheraccumulations of damage (2). (ii) Certain growth conditions(e.g., 30°C with addition of 0.05% glucose) led to lowerlevels of endonuclease V accumulation as detected on West-ern blots without affecting UV resistance as measured bycell survival. (However, further decreases in endonucleaseV levels, e.g., with 0.4% glucose addition, resulted in acorresponding loss of UV resistance.)Although the amount of endonuclease V found within

these cells is sufficient for substantial UV repair, maximalaccumulations are only about 0.2% of total cellular proteins.This moderately low level of plasmid-encoded protein prob-ably reflects proteolytic degradation of this expression prod-uct. Such a rationale is supported by the extreme sensitivityof endonuclease V production to elevated culture tempera-tures which normally are well within physiologic limits (Fig.3). Thus, it appears that the enzyme expressed from theseconstructions may be somewhat labile and potentially tar-geted for degradation. The temperature sensitivity of endo-nuclease V production was further corroborated by a phageM13mpl8 clone in which the oIPR-denV sequence wasinserted in the polylinker cloning region. Infections ofE. coliUT481 at 37°C with this phage demonstrated no im-munoreactive endonuclease V accumulation in infectedcells. In contrast, when these infections were initiated at37°C (to ensure efficient adsorption) but continued at 27°C,moderate levels of immunoreactive endonuclease V were

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VOL. 168, 1986

TABLE 1. Bacterial strains, plasmids, and bacteriophage

Strain, plasmid,Strain,p 'asmid, Genotype or phenotype Sourceor phageE. coliUT481 met thy A(lac-pro) hsdRBamHI hsdM+ supD C. Lark

TnlO/F' traD36 proAB lacIqZAM15GX1200 A(lac-pro) supE thi nadA: :TnlO(X cI ts857 Genex Corp.

ABamHI) A(chlD-blu)4 galKlF' traD36 proABlacIqZAM15

AB2480 uvrA6 recA13 arg+a A. GanesanAB2487 recA13 thyA16 drm-1a A. GanesanGW5837 hflA: :TnS lon: :TnlO lexA sulAa G. Walker (I. Herskowitz,

S. Gottesman)GW5838 css-l::TnS lexA sulAa G. WalkerGW5844 css-7: :TnS lexA sulAa G. WalkerGW5845 css-12: :TnS lexA sulAa G. WalkerGW5847 css-14::TnS lexA sulAa G. WalkerGW5858 css-25: :TnS lexA sulAa G. WalkerGW5959 css-26 lexA sulAa G. WalkerGW5865 css-32 lexA sulAa G. WalkerB Wild type (sulA lon) G. Mosig

PlasmidpGX2608 Apr A OLpR A t4s GalK+ Genex Corp.pGX2608-16-denV+ Apr A OLPR endonuclease VI A t4S GalK+ This laboratorypGX2608-19-denV Apr X OLPR endonuclease V-b A t4s GalK+ This laboratorypGX2608-22-denV+ Apr X OLPR endonuclease V+(Ile)c X t4s GalK+ This studypGX2608-23-denV Apr A OLPR endonuclease V- (Ile)b,c X t4s GalK+ This study

PhageM13mp8vn'5 Lac' (mp8: :350-bp Xgtll-IIv 1 MspI fragment)d This laboratoryM13mp9v,+3 Lac' (mp9: :750-bp Xgt11-IIvj 6 MspI fragment)' This laboratoryT4D Wild type A. GanesanT4v1 denV A. Ganesana These strains additionally contain the E. coli AB1157 genetic markers thr-1 leu-6 thi-1 supE44 lacY) galK2 ara-14 xyl-S mtl-1 proA2 his-4 argE3 str-31 tsx-33

sup-37 (1).b The endonuclease V gene is inserted in the reverse orientation relative to the OLPR promoter (see the text).c Two isoleucine codons in the endonuclease V coding sequence have been altered to synonym triplets (see the text).d This insert contains the 5' EcoRI fragment of T4 denV (Lloyd and Augustine, in press).eThis insert contains the 3' EcoRI fragment of T4 denV (8).

identified in lysates of infected cells. Here, in a differentgenetic background, the extent of accumulation of endonu-clease V was again very sensitive to growth temperature.Recently, Chenevert et al. (4) have reported the expressionof endonuclease V from the tac promoter. These investiga-tors found that, after isopropyl-,-D-thiogalactopyranosideinduction of the tac-denV plasmid, endonuclease V accumu-lated to -40% of total cellular proteins. They observed thatthis level of endonuclease V accumulation correspondedwith lethality to the E. coli host. Since the levels at whichour denV constructions accumulated endonuclease V wereconsiderably lower than that noted above, we concluded thatenzyme accumulation of 0.2% of total cellular protein is notlethal. The differences in the levels of accumulation may

simply reflect different efficiencies in translation initiationwith the tac-denV mRNA versus the PR-denV mRNA, sinceboth tac and PR are strong inducible promoters. If translationinitiation is more efficient from tac-denV mRNA, the in-creased rate of protein synthesis may overcome the proteindegradation problem which we encountered with our con-struction.

Presently, milligram quantities of endonuclease V, puri-fied to homogeneity, are routinely isolated from E. coliAB2480 cultures harboring plasmid pGX2608-16-denV+ (K.Higgins and R. Lloyd, Mutat. Res. in press). These enzymeisolates are being used in studies to further elucidate theinteraction of endonuclease V with DNA bearing UV-induced lesions.

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ECLi2

D iu\ uvrA-recA-w/6 _ pGX2608-16(X t (denV+)

uvrArec A-w/0,1- pGX2608-16

_ \(denVi)+glucose

uvrA7recA-w/pGX2608 -19

(denVi

.F1 ,2 3 4UV, J/m2

FIG. 4. Effect of glucose on accumulation of endonuclease V andon UV resistance in AB2480(pGX2608-16 [denV+]). Inset: Westernblot detection of endonuclease V accumulated in stationary-phasecells grown at 30°C in LB media supplemented with various con-

centrations of glucose (A, lanes 2 to 9) and glycerol (B, lanes 9 to17). The final concentrations of supplemented glucose or glycerolwere 0, 0.01, 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8%. Lane 1, partiallypurified preparation of endonuclease V from T4-infected E. coli. Thearrowhead indicates the position of endonuclease V. The bands ofM, standards were as indicated (in thousands). Colony-formingability of UV-irradiated stationary-phase cells grown at 30°C.AB2480 harboring plasmid constructs as indicated were grown in (Oand *) LB medium or (0) LB medium-0.4% glucose.

We thank Graham Walker and Chris Dykstra for use of their lacZstabilization strains and for their helpful comments concerningendonuclease V turnover. We also extend our appreciation to AnnGanesan and to members of the Genex Corporation, Gaithersburg,Md., for supplying us with strains, plasmids, and phage used in thiswork. Special thanks is given to Doris Harris for her patience andhours of labor on the manuscript.

This research was supported by Public Health Service grant ES00267 from the National Institutes of Health.

LITERATURE CITED

1. Bachmann, B. J. 1972. Pedigrees of some mutant strains ofEscherichia coli K-12. Bacteriol. Rev. 36:525-557.

2. Bernstein, C., and S. S. Wallace. 1983. DNA repair, p. 138-151.In C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget(ed.), Bacteriophage T4. American Society for Microbiology,Washington, D.C.

3. Burnette, W. N. 1981. "Western blotting": electrophoretictransfer of proteins from sodium dodecyl sulfate-polyacryl-amide gels to unmodified nitrocellulose and radiographic detec-tion with antibody and radioiodinated protein A. Anal.Biochem. 112:195-203.

4. Chenevert, J. M., L. Naumovski, R. A. Schultz, and E. C.Freidberg. 1986. Partial complementation of the UV sensitivityof E. coli and yeast excision repair mutants by the cloned denVgene of bacteriophage T4. Mol. Gen. Genet. 203:163-171.

5. Friedberg, E. C. 1975. Review article: DNA repair of ultra-violet-irradiated bacteriophage T4. Photochem. Photobiol.21:277-289.

6. Gordon, L. K., and W. A. Haseltine. 1980. Comparison of thecleavage of pyrimidine dimers by the bacteriophage T4 andMicrococcus luteus UV-specific endonucleases. J. Biol. Chem.255:12047-12050.

7. Grantham, R., C. Gautier, M. Gouy, M. Jacobzone, and R.Mercier. 1981. Codon catalog usage is a genome strategymodulated for gene expressivity. Nucleic Acids Res. 9:r43-r74.

8. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

9. Lloyd, R. S., and M. L. Augustine. 1986. Cloning and expressionof the 3' portion of the T4 denV gene as a lacZ fusion gene.Mutat. Res. 165:89-100.

10. Lloyd, R. S., and P. C. Hanawalt. 1981. Expression of the denVgene of bacteriophage T4 cloned in Escherichia coli. Proc. Natl.Acad. Sci. USA 78:2796-2800.

11. McMillan, S., H. J. Edenberg, E. H. Radany, R. C. Friedberg,and E. C. Friedberg. 1981. denV gene of bacteriophage T4 codesfor both pyrimidine dimer-DNA glycosylase and apyrimidinicendonuclease activities. J. Virol. 40:211-223.

12. Nakabeppu, Y., and M. Sekiguchi. 1981. Physical association ofpyrimidine dimer DNA glycosylase and apurinic/apyrimidinicDNA endonuclease essential for repair of ultraviolet-damagedDNA. Proc. Natl. Acad. Sci. USA 78:2742-2746.

13. Nakabeppu, Y., K. Yamashita, and M. Sekiguchi. 1982. Purifi-cation and characterization of normal and mutant forms of T4endonuclease V. J. Biol. Chem. 257:2556-2562.

14. Radany, E. H., and E. C. Friedberg. 1980. A pyrimidinedimer-DNA glycosylase activity associated with the v geneproduct of bacteriophage T4. Nature (London) 286:182-185.

15. Radany, E. H., L. Naumovski, J. D. Love, K. A. Gutekunst,D. H. Hall, and E. C. Friedberg. 1984. Physical mapping andcomplete nucleotide sequence of the denV gene of bacterio-phage T4. J. Virol. 52:846-856.

15a.Recinos, A., and R. S. Lloyd. 1986. Efficient screening ofrecombinant DNA junctions afforded by probing with synthetic"bridge" oligonucleotides. Biochem. Biophys. Res. Commun.138:945-952.

16. Seaweil, P. C., C. A. Smith, and A. K. Ganesan. 1980. denV geneof bacteriophage T4 determines a DNA glycosylase specific forpyrimidine dimers in DNA. J. Virol. 35:790-797.

17. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

18. Valerie, K., E. E. Henderson, and J. K. deRiel. 1984. Identifi-cation, physical map location and sequence of the denV genefrom bacteriophage T4. Nucleic Acids Res. 12:8085-8096.

19. Valerie, K., E. E. Henderson, and J. K. deReil. 1985. Expressionof a cloned denV gene of bacteriophage T4 in Escherichia coli.Proc. Natl. Acad. Sci. USA 82:4763-4767.

20. Warner, H. R., L. M. Christensen, and M.-L. Persson. 1981.Evidence that the UV endonuclease activity induced by bac-teriophage T4 contains both pyrimidine dimer-DNA glycosylaseand apyprimidinic/apurinic endonuclease activities in the en-zyme molecule. J. Virol. 40:204-210.

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