Proc. Nati. Acad. Sci. USAVol. 77, No. 5, pp. 2796-2800, May 1980Genetics

Gene-directed mutagenesis in bacteriophage T7 provided bypolyalkylating RNAs complementary to selected DNA sites

(DNA-RNA crosslinking reagent/R loops/local DNA modification)

RUDOLF I. SALGANIK*, GRIGORIY L. DIANOV*, LYUDMILA P. OVCHINNIKOVAt, ELENA N. VORONINA*,ELENA B. KOKOZAt, AND ALEXANDER V. MAZIN**Institute of Cytology and Genetics, Siberian Division of the U.S.S.R. Academy of Sciences; and tNovosibirsk State University, 630090 Novosibirsk, U.S.S.R.

Communicated by W. A. Engelhardt, January 3,1980

ABSTRACT Bacteriophage 17 early transcripts were usedas carriers of alkylating groups to affect complementary T7DNA sites for inducing mutations in preselected genes. A het-erofunctional polyalkylating agent NNN'-trik(-chloroethyl-N'jformylphenyl)propylene diamine-1,3 was attached to3-5% of the transcript nucleotides. The controlled alkylatinggroups carried on RNA were activated after RNA-DNA hy-bridization. The modified transcripts were shown to hybridizeonly with the complementary H strand and to form covalentlybound R loops in the appropriate 17 sites. The ¶7 DNA mole-cules locally alkylated by the modified transcripts of gene 1.3coding for T7 ligase were packaged into ¶7 proteins and usedto infect Escherichia coli B. As judged by plating efficiency onthe ligase-deficient E. coil BL2 strain, 4 of 140 plaques obtainedafter infection contained mutants defective in gene 1.3 inamounts of 0.7-1.5%. The ¶7 DNA locally' alkylated by themodified transcripts of genes 0.3 and 1.1 were used for thetransfection of E. coli C1757. Analysis of 24 plaques produced.by transfection indicated that 3 contained mutants defectivein gene 0.3 in amounts of 2-10%. The mutants had alio a secondunidentified mutation. Complementation analysis data suggestthat the second mutation is due to a defect in geneI1.1. The re-sults obtained demonstrate the efficiency of the approach de-veloped for gene-directed mutagenesis.

It has been shown (1) in this laboratory that polynucleotidescarrying a number of artificially attached highly reactivegroups, capable of modifying deoxynucleotide residues, canaffect definite continuous stretches of complementary DNA.The aim of this site-directed modification of DNA was to inducemutations in selected genes or complete inactivation of alienDNA or RNA. A general scheme of this approach is given inFig. 1.The appropriate experimental conditions for this work have

been specified (1-4). For chemical DNA modification, a specialpolyfunctional nitrogen mustard N,N,N'-tri-(f3-chloroethyl)-N'-(p-formylphenyl)propylene diamine-1,3 (TFP) with con-trolled reactivity was synthesized:

,s ~ ~ <CH2CH2CI

OHC \N-CH2CH22-NCH2CH2C1

vH2

tHI

The highly reactive aliphatic (f3-chloroethyl)amino group(Ri) of the compound was used to attach the reagent to thepolynucleotide complementary to the selected DNA site,thereby transforming the polynucleotide into an addressed

a A B C

DNA 1111111II I

I I IM&I A 2 -RNA

b

I

A B C

c

FIG. 1. Schematic representation of gene-directed matagenesis.(a) RNAs transcribed from genes A, B, and C. (b) Attachment ofhighly reactive chemical groups to the transcript of gene B. (c)Chemical modification of gene B with the corresponding transcript,carrying highly reactive groups, after DNA-RNA hybridization.

carrier of potentially active aromatic alkylating groups (R2).The reactivity of the R2 groups is strongly inhibited by theneighboring formyl residues (5). However, the R2 groups canbe activated, when needed-for instance, after the hybridiza-tion of modified RNA to the complementary DNA. Reductionof the formyl residue by sodium borohydride markedly in-creases the reactivity of the R2 groups (6). The use of comple-mentary RNA molecules carrying such controlled alkylatinggroups makes DNA modification highly site-directed becauserandom alkylation of DNA sites beyond the complementaryregion becomes virtually impossible.

This paper describes experiments in which specific polyal-kylating T7 early RNAs were used to induce site-directedmutations in mapped genes of the bacteriophage T7.

MATERIALS AND METHODS

H and L strands of T7 DNA were separated according to Szy-balski et al. (7). The total early T7 transcripts were preparedby transcription of T7 DNA with E. coli RNA polymerase(8).The T7 early RNAs were extracted from T7-infected E. coli,

separated by polyacrylamide gel electrophoresis, and identifiedaccording to Studier (9). Attachment of the alkylating reagentTFP to the RNA was carried out in 50% methanol/5 mMTris-HCI, pH 7.5, at room temperature for 10-15 min. The

Abbreviation: TFP, N,N,N'-tri-(f3-chloroethyl)-N'-(p-formylphe-nyl)propylene diamine-1,3.

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reaction mixture contained 0.8 mM TFP and 50-100 Ag ofRNA per ml. The alkylated RNA was precipitated with ethanol(2 hr at 20°C) and centrifuged; the pellet was resuspended in10 mM Tris-HCl (pH 7.5). To remove the unbound reagent, theRNA solution was applied to a Sephadex G-50 column equili-brated and eluted with 10mM Tris-HCl (pH 7.5). To estimatethe percentage of the modified nucleotides in TFP-treatedRNA, the latter was hydrolyzed (1 M NaOH, 18 hr, 37°C) andthe modified and nonmodified nucleotides were analyzed bypaper chromatography (3). The TFP was synthesized by A. A.Gall, C. V. Shishkin, and V. A. Kurbatov; the details of theprocedure appear elsewhere (6). The hybridization of thepolyalkylated early T7 RNAs to T7 DNA and the formation ofR loops are described in the legend to Fig. 3.

After the formation of R loops, the RNA-borne alkylatinggroups were activated by the addition of sodium borohydrideto the solution to a final concentration of 10 mM; the localintra-complex alkylation of T7 DNA was then carried out for4 hr at 40°C. T7 DNA with covalently bound T7 early tran-scripts was packaged into T7 coat proteins (10), and the re-

assembled bacteriophage particles were used for the infectionof E. coli. In another experimental series, locally alkylated T7DNA was directly used for the transformation of E. coil (11).Isolation of bacteriophage T7 mutants, plating, and comple-mentation tests were performed according to Studier (12).The various B and C strains of E. coli used for testing the T7

mutants were kindly provided by F. W. Studier.

RESULTSDuring the transcription of the early region of T7 DNA by E.coli RNA polymerase, synthesis proceeds asymmetrically withonly the H strand of T7 DNA being transcribed (13). Accord-ingly, the total T7 early transcripts hybridize with H strandsand do not hybridize with the L strands of T7 DNA. Eventhough modified by attachment of TFP to 4-5% of the nitrogenbase residues, the T7 early transcripts retained their ability tointeract only with the complementary H strands (Fig. 2).

100

80 -

.2~~~~~~~~

60

Vn0 20 30 40 50

Time, min

FIG. 2. Kinetics of hybridization of the H and L strands of T7DNA with intact or modified early T7 3H-labeled transcripts. Totalearly T7 3H-labeled transcripts were modified by TFP to the extentof 5-6%. Hybridization was carried out in 0.3 M NaCl/0.03 M sodiumcitrate at 65°C; the concentrations of DNA and transcripts were 1pg/ml. Aliquots of the reaction mixture were taken every 5-10 minand treated with RNase (40 ,g/ml), 1 hr at room temperature). Thesamples were applied on Whatman 3 MM filters, washed with 5%trichloroacetic acid, ethanol, and ether, and assayed for radioactivity.Curves: 1, H strand + intact transcript; 2, H strand + modified; 3, Lstrand + intact transcript; 4, L strand + modified transcript.

The T7 early transcripts, carrying the potentially active R2alkylating groups, were used for the alkylation of the comple-mentary T7 DNA sites. For this purpose, the modified tran-scripts were hybridized with T7 DNA, and R loops wereformed. After completion of hybridization, the R2 groups wereactivated by reduction of the neighboring formyl residues. Itwas expected that intra-complex alkylation of DNA wouldresult in covalent binding of the polyalkylating transcript toDNA. The procedure would make the complex resistant todenaturation conditions. To ascertain that DNA was alkylatedwithin the DNA-RNA complex, the stability of the complex wastested. It is known that RNA becomes insensitive to RNasehydrolysis when complexed with DNA. Hybridization with T7DNA rendered the unmodified 32P-labeled transcripts resistantto RNase, but the transcripts became susceptible-to the enzymichydrolytic action after denaturation (Table 1). Polyalkylating.32P-labeled transcripts were hybridized with T7 DNA and theirR2 alkylating groups were activated. In spite of denaturation,the transcripts remained resistant to RNase. This indicated that,as a result of alkylation, the transcripts were covalently boundto T7 DNA.To test whether T7 DNA alkylation was restricted to a spe-

cific complementary site, the R loops formed by the polyal-kylating covalently bound T7 early transcripts in the T7 DNAwere located by electron microscopy. Judging by electron mi-croscopy data, intact T7 early transcripts formed R loops pre-dominantly at one end of the T7 DNA molecules and occupiedabout 20% of the total T7 DNA length. This agrees with theprevious data on T7 early gene location (14). The R loopsformed by the polyalkylating T7 transcripts covalently boundto the T7 DNA were localized in the same position as the intacttranscripts (Fig. 3). Obviously, the polyalkylating T7 transcriptsalkylate the complementary T7 DNA sites.To apply this approach to gene-directed mutagenesis, indi-

vidual T7 RNA molecules transcribed in vivo from early T7genes were prepared. In vivo 32P-labeled early transcripts ofbacteriophage T7 were separated by gel electrophoresis, locatedby autoradiography, and identified as described by Studier (9).Fig. 4 presents a typical electrophoretic pattern of 32P-labeledT7 early transcripts. The transcripts of early gene 1.3 and amixture of early gene 0.3 and 1.1 transcripts were selected. Thefunction of gene 1.3 is to code T7 DNA ligase (17). One of themain functions of gene 0.3 is to overcome host restriction (18).The function of gene 1.1 is still unknown.

Table 1. Effect of denaturation on T7 DNA with R loops formedby intact or polyalkylating T7 32P-labeled transcripts after

intracomplex DNA alkylation

Radioactivity ofT7 DNA-32P-labeled

transcript complex, cpmTranscript Before After

in T7 DNA R loop denaturation denaturation

Unmodified 3600 20Polyalkylating 3060 2664

T7 DNA with R loops was prepared as described in the legend toFig. 3. After activation of the RNA-borne alkylating groups (by theaddition of sodium borohydride) and intra-complex DNA alkylation,formamide concentration was increased to 90% and R loops weredenatured by incubation at 50'C for 30 min; an equal volume of 0.5M NaCl/1 mM EDTA/10 mM Tris*HCl, pH 7.5, containing RNases(RNase A, 20 ,ug/ml; RNase T1, 20 units/ml) was added and themixture was incubated for 1 hr at room temperature. The treatedsamples were then cooled on ice, and an equal volume of 1% N-cetyl-N,N,N-trimethylammonium bromide solution was added. Theprecipitates were collected on GF/C filters (Whatman) and washedwith 50 ml of 0.01% N-cetyl-N,N,N-trimethylammonium bromide.

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N.7~~~ ~ ~~~~~~~~A-X .....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

S~~~~~~~~~\C

.j.

N-"

SMA.6i~~~~~~~~~~~~~~~~~~~~~*~

FI3ElctonmirgrphofT7DN wt a Rlop ore b te olalyltg ary 7 NARlopswee oredi 7% oramdelmMET/ iprzn i 2ehnsloi ai nwihte oa ocnrtino ain as01 h ecto a areoufr6rt8Teonenraios f NAan RA n hesoutonwee 0 igml(o ech (5)T7DN smpeswee pradfo eecromicroscopy ~ 14byteKencmd oraietcnqe(6

The individual T7 RNA fractions were extracted from thegels by electroelution and deproteinized with phenol. A 3-4%modification of the purified RNA was achieved by alkylationwith TFP R loops were obtained by hybridization of TFP-

Gene

FIG. 4. Electrophoretic patterns of T7early RNAs. Early T7 RNAs were labeled

0.7 with 32p in the presence of chloramphenicolbetween 0 and 10 min after infection of UV-irradiated E. coli B (9). The cells were col-

-1.3 lected by centrifugation (5000 X g, 20 min)and lysed in a small volume of 1% NaDod-SOJ1% 2-mercaptoethanol/1 mM EDTA/50mM Tris-HCl, pH 7.5, for 2 min at 85°C.Electrophoresis in 2.25% acrylamide/0.5%

0.3; 1 .1 agarose gel was carried out for 3 hr at 70 V.Gene functions: 1, T7 RNA polymerase; 0.7,protein kinase; 1.3, T7 ligase; 0.3, overcomes

Kt2 host restriction; 1.1, unknown.

modified RNA with T7 DNA. The R2 groups were then acti-vated by reduction of formyl residues with sodium borohydride.The T7 DNA molecules, which had been locally alkylated bythe modified transcripts of gene 1.3, were packaged in vitro intoT7 phage particles. E. coli B was infected with these assembledparticles.Each plaque obtained after infection of E. coli B with re-

constituted phage particles produced after replating was testedfor ability to grow on the ligase-deficient E. coli BL2 strain. Asjudged by plating efficiency on E. coli BL2, 4 of 140 plaquescontained T7 mutants defective in gene 1.3. Because only onestrand of the transfected T7 DNA was modified, each of theseplaques contained the progeny of the intact and modified T7DNA strands. Hence, only the modified T7 DNA strand pro-duced phage mutants. The amounts of mutant phages foundin each of the four plaques varied from 0.7 to 1.5%. In controlexperiments, T7 DNA with R loops formed by the intact RNAof gene 1.3 was used for the in vitro packaging and productionof phage particles. E. coli B was infected with assembled pha-ges; 100 plaques were tested further by plating on E. coli BL2and no gene 1.3 mutants were detected.T7 DNA in which genes 0.3 and 1.1 had been locally alkyl-

ated by appropriate TFP-modified transcripts was used totransfect permissive E. coli C1757. Although possessing sup-pressor activities (Sup D), this strain lacks a restriction system(hsp 0). The mutants were tested on E. coli B, which has a re-striction system but no amber suppressors, and on E. coli C,which has no restriction system and no amber suppressors (18).

2798 Genetics: Salganik et al.

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Table 2. Relative plating efficiency of T7 phage mutants obtained by E. coli transfection withDNA locally alkylated-with transcripts of genes 0.3 and 1.1

Relative plating efficiency at 370CtMutants Mutants Mutants

E. coli Amber Wild from from fromstrain suppressor hsp* type plaque I plaque II plaque III

C1757 Sup D 0 1.0 1.0 1.0 1.0C None 0 0.8 1.2 x 10-5 0.1 X 10-4 0.15 X 10-4B None B 1.2 1.3 X 10-5 0.1 X 10-4 0.1 X 10-4

*The hsp locus is responsible for host restriction and modification; 0, the strain neither restricts normodifies; B, the strain is capable of restricting and modifying with B specificity (19).

t Plating efficiency estimates were based on growth on E. coli C1757.

This test makes it possible to identify mutants defective in gene0.3 and amber mutants of each essential gene. Analysis of thebehavior of 24 plaques produced by transfection indicated that3 contained mutants with low plating efficiency on E. coli B(Table 2). The amount of mutant phages contained by each ofthree plaques varied from 2 to 10%. However, the plating ef-ficiency of these mutants also was decreased on E. coli C. Thissuggested that, besides a defect in gene 0.3, the mutants had a

secondary mutation affecting their plating ability on E. coliC.

Inasmuch as mutations were produced by the local alkylationof genes 0.3 and 1.1, it was reasonable to assume that both geneswere affected. However, it could not be ruled out that anotherunknown gene was responsible for the simultaneous decreasein the plating efficiency of mutants on E. coli B and C. Addi-tional evidence was needed to demonstrate independently theinvolvement of at least gene 0.3. By recombination with wild-type bacteriophage T7, the secondary unidentified mutationwas removed and, after that, only the mutation in gene 0.3 re-mained. As shown in Table 3, the mutants obtained plated wellon E. coli C but poorly on the restrictive E. coli B. The mutantswere temperature sensitive. Similar mutations of gene 0.3 havebeen described by Studier (18).

According to complementation data, all mutants from thethree initial plaques could be assigned to one complementationgroup. Hence, the unidentified mutation in all three clones didnot arise randomly; rather, it resulted from an alteration at thesame locus. Our experiments demonstrated that the mutantsof the three clones are not members of the complementationgroups of genes 1.0 and 1.3; this increases the probability thatthe unidentified mutation is a defect of gene 1.1.

DISCUSSIONThe present report provides evidence that RNA moleculescarrying highly reactive groups are capable of alkylating locallycomplementary DNA sites. It has been shown (2) previously inthis laboratory that, when the alkylating agents are attachedto 10% of the RNA bases, the polynucleotide loses its capacityto hybridize with the complementary DNA strand. When 5-6%of the RNA bases are modified, the RNA-DNA complex is im-perfect in that a part of the RNA is sensitive to RNase; at amodification level of 3-4%, the RNA in the RNA-DNA com-

plementary complex is resistant to RNase (2). Clearly, thismodification level of the T7 transcripts provides optimal con-ditions for the interaction with DNA target sites. It is note-worthy that the RNA molecules carrying activated alkylatinggroups interact only with the complementary DNA strand andalkylate it; they do not alkylate the noncomplementary one,even though reaction conditions may be optimal. This is whatoccurs during the interaction of the polyalkylating early T7transcript with the complementary T7 DNA H strand. It ap-pears that polynucleotide molecules have to be brought closetogether to ensure alkylation of DNA. Alkylation efficiency is

very low outside the complex. Thus, the noncomplementaryL strand is not alkylated by the modified T7 transcript, althoughthe alkylating groups attached to the transcript are activated(1). Random collisions of large linear molecules presumablyhinder their interaction.The results obtained demonstrate that mutations are induced

only in particular genes, those chosen beforehand and whoseRNA transcripts were used for alkylation of T7 DNA. Thenumber of induced mutations was unusually high. In the caseof gene 0.3, it amounted to 12%, and we did not find mutationsin any other gene, although the method used for identificationof gene 0.3 mutants is sensitive enough to disclose an ambermutation of any essential gene of bacteriophage T7. Thenumber of induced mutations in gene 1.3 was almost 3%.How do the polyalkylating RNA transcripts exert their mu-

tagenic action? If, upon entry into a bacterial cell, T7 DNAretains the covalently attached transcript, this would deprivethe H strand of T7 DNA of its replicative capacity. No muta-tions would then arise because only the unaffected L strandwould replicate. It seems more likely that the transcript, co-valently bound to T7 DNA, may be hydrolyzed by an enzymeof RNase H type; if so, the modified H strand would retain onlythe ribonucleotide residues covalently bound through TFP tothe deoxynucleotides, which may be lost easily by the H strand(19). During subsequent replication, substitutions of thetransversion and transition types may occur.The addressed modification of DNA by complementary

oligonucleotides carrying an alkylating group at the 3' end wasproposed by Grineva and associates (19, 20) and applied tosite-directed nicking of DNA (21). Later, we (1), and inde-pendently Summerton and Bartlett (22), suggested modifyinglong stretches of DNA by complementary polynucleotidescarrying numerous reactive groups. Some current approachesto the site-directed mutagenesis are based on the modificationof mutagen-sensitive one-stranded regions of DNA producedin the restriction sites (23, 24). The method we developed is freeof limitations imposed by the predetermined localization ofendonuclease restriction sites.

Hopefully, this approach may be extended to the productionof mutants by the inactivation of definite genes or even entirealien genomes. Individual mRNA or their definite parts, DNA

Table 3. Relative plating efficiency of T7 phage mutantsin gene 0.3

Relative plating efficiencyAt 230C At370CPlaques Plaqueswith with

E. coli wild-type wild-type Small-sizedstrain morphology morphology plaques

C 1.0 1.0 0B 0.22 0.19 x 10-2 0.05

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restriction fragments, and synthetic oligo- and polynucleotides,can be utilized as carriers of highly reactive groups.

The authors express their gratitude to Prof. D. G. Knorre and Dr.M. A. Grachev for helpful discussions. They also thank Drs. V. N.Zaytsev and V. I. Vavilin for assistance in the electron microscopystudies.

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2. Dianov, G. L., Bondar, T. S. & Salganik, R. I. (1979) Mol. Biol.(USSR) 13, 383-387.

3. Salganik, R. I., Dianov, G. L., Kokoza, E. B., Ovchinnikova, L.P., Kurbatov, V. A., Mustaev, A. A., Gall, A. A. & Shishkin, G. L.(1979) Mel. Biol. (USSR) 13,625-632.

4. Dianov, G. L., Mazin, A. V., Zaytsev, B. N., Vavilin, V. I. & Sal-ganik, R. I. (1980) Mol. Biol. (USSR) 14, in press.

5. Belikova, A. M., Vakhrusheva, T. E., Vlasov, V. V., Grineva, N.I., Kurbatov, V. A. & Knorre, D. G. (1969) Mol. Biol (USSR) 3,210-220.

6. Gall, A. A., Kurbatov, A. A., Mustaev, A. A. & Shishkin, G. V.(1979) Izv. Sib. Otd. Akad. Nauk SSSR, Ser. Khim. Nauk 2,99-104.

7. Szybalski, W., Kubinski, H., Hradecna, Z. & Summers, W. C.(1971) Methods Enzymol. 210,383-413.

8. Studier, F. W. (1973) Proc. Nati. Acad. Sci. USA 70, 1559-1563.

9. Studier, F. W. (1973) J. Mol. Biol. 79,237-248.10. Kerr, C. & Sadowski, P. D. (1974) Proc. Nati. Acad. Sci. USA 71,

3545-3549.11. Mandel, M. & Higa, A. (1970) J. Mol. Biol. 53, 159-162.12. Studier, F. W. (1969) Virology 39,562-574.13. Minkley, E. G. (1974) J. Mol. Biol. 83,305-315.14. Simon, M. N. & Studier, F. W. (1973) J. Mol. Biol. 79, 249-

265.15. Thomas, M., White, R. L. & Davis, R. W. (1976) Proc. Nati. Acad.

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502-508.20. Belikova, A. M., Zarytova, V. F. & Grineva, N. I. (1967) Tetra-

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