Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA.

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0022-538X/79/02-0529/07$02.00/0

Involvement of

DNA

Gyrase

in

Replication

and

Transcription

of Bacteriophage T7

DNA

MARK A.DR WYNGAERTANDDAVIDC. HINKLE*

Departmentof Biology, Universityof Rochester, Rochester,NewYork 14627

Received forpublication21September1978

GrowthofbacteriophageT7 is inhibitedbythe antibioticcoumermycin A1,an

inhibitor of the Escherichia coli DNA gyrase. Since growth of the phage is

insensitive to the antibiotic in strains containingacoumermycin-resistant DNA gyrase,this enzyme appearstoberequiredforphage growth.We haveinvestigated the effectofcoumernycinonthe kinetics ofDNA, RNA, andprotein synthesis during T7 infection. DNAsynthesis is completelyinhibitedbythe antibiotic. In

addition, coumermycin significantly inhibits transcription of late butnot early

genes.Thus,E. coli DNAgyrase mayplayanimportantrole intranscriptionas

wellas inreplicationof T7 DNA.

The Escherichia coli enzyme DNA gyrase introducesnegative superhelicalturnsintoa cir-cular,double-strandedDNA in an ATP-depend-ent reaction. Thegenetic locus(cou)which

de-termines resistance to coumermycin A, and to

novobiocin has beenidentified ascontrollingthe

activityof DNA gyrase(5).DNAgyrase isolated from wild-type cellsis inhibited byboth these

antibiotics, whileenzyme from a Cour strain is

resistanttoinhibition. Morerecently, asecond subunit of DNA gyrase has been identified as

the product ofnalA, a different genetic locus

which determines resistance to nalidixic acid and to oxolinic acid (22). Thus, DNA gyrase appears to be the specific targetfor inhibition

byall four of these antibiotics.

It has beenknown for sometimethat nalidixic

acid inhibitsthereplicationofbacteriophageT7

DNA (1). Therecentidentificationof thenalA

protein as a subunit of DNA gyrase suggests that thisnovel enzymeplaysanessential rolein

T7DNAreplication. Consistent withthis, Itoh and Tomizawa have recently shown that T7

DNA replication is also sensitive to the anti-bioticcoumermycin (8).

We have investigated the effect of

coumer-mycinonbacteriophageT7nucleic acid

synthe-sis.Ourresults indicate that E. coliDNAgyrase isrequiredfor efficienttranscriptionofmanyof

thelatephagegenes, aswellasforphageDNA

synthesis.

MATERIALS AND METHODS

Bacterial strains and bacteriophage. The E. coli K-12 strains NI748(coumermycin resistant) and

NI 747 (coumermycin sensitive), which are parallel

transductantsofCRT46 (5), were obtained from M.

Gellert.E.coliB/1wasfrom M.Chamberlin.T7

wild-typeandamber mutants were obtained from F. W.

Studier.For convenience, T7 mutants are designated

bygeneonly. The amber mutations used are gene 1,

am193; gene 5, am 28; gene 6, am 147.

Chemicals. Coumermycin Al (referredto as

cou-mermycin), lot 448, was a gift of W. F. Minor, Bristol

Laboratories, Syracuse,N.Y.Coumermycin wasadded

tocultures from a stock solution (10 mg/ml) in

di-methyl sulfoxide. [3H]thymidine (20 Ci/mmol) and

[3Hluridine (20 Ci/mmol) were from New England NuclearCorp.,and"4C-labeledL-amino acid mixture (50mCi/mAtom of carbon) was from Amersham.

Kinetics ofDNA, RNA,andproteinsynthesis.

Bacteria were grown in a gyratory shaker at

300C

in M9 medium or, for the K-12 strains NI 747 and NI 748, in M9 mediumsupplemented,perml, with 2 ,ug of

thymine,5

jug

ofthiamine,and60,ugeachof threonine,

valine, leucine,and isoleucine. At a density of 2.5 x

10'cellsperml,bacteria wereinfectedwith T7phage

at amultiplicityof 10. To measure the rate of DNA,

RNA, or protein synthesis, 0.1-mlsamples were

re-movedfromthe culture andplacedintubescontaining

10p1 of[3H]thymidine (50 ,uCi/ml), [3H]uridine (50

MCi/ml),or

"4C-labeled

amino acids (5

ACi/ml).

After 1min at300C,synthesis was terminated by the addi-tion of 3 ml of cold 10% trichloroacetic acid. The

precipitateswerecollectedonWhatmanGF/C filters

andwashed five times with 3 ml of cold 1 N HCI and

finallywith 3 ml of coldethanol. Radioactivityonthe dried filters was determined in atoluene-basedsolvent

in aliquidscintillationcounter.

UVirradiation. Forexperimentsin whichbacteria wereirradiated with UV light prior to T7 infection, 10

mlofbacteria(2.5x10'cellsper ml) was placed in a

sterileopenpetridish(12 by 100 mm) andirradiated at 254 nmwith a GeneralElectric G8T5germicidal lampfor 2.5 min at a fluence of 3.3J/m2per s. The

cells were thenreturnedtoa sterile flask and

incu-bated in a gyratory shaker at

300C

for 20minprior to infection.

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530 DE WYNGAERT AND HINKLE

RESULTS

Coumermycin inhibits growth of bacte-riophage T7. Theeffect ofcoumermycinonthe growth of phage T7 in E. coliB/1 and in the isogenic K-12 strains NI 747 Cou' and NI 748 Courisshown inFig.1.In10-,ug/ml coumermy-cin, the burst sizeinB/1 wasreducedto6% of the control. Growth in both K-12 strains was

somewhatless sensitive to the drug, but at 10

,ug/ml

the burst in the coumermycin-sensitive

strainwasreducedto20%ofthecontrol,whereas growth in the resistant strain was unaffected. Since the coumermycin-resistant strain NI 748 has been shown to contain a coumermycin-re-sistant DNA gyrase (5), these results suggest thattheE. coliDNA gyraseplaysan essential roleinbacteriophage growth.

Effectof coumermycinonT7DNA,RNA,

and

protein synthesis.

The effect of

coumer-mycin on DNA, RNA, and protein synthesis duringT7 infection ofE. coli B/1 isshown in Fig. 2a, b, and c. The rate of both DNA and RNAsynthesiswassignificantlyreduced in the presenceof the antibiotic. Proteinsynthesiswas

alsoreduced, butthe effectwaslesspronounced and probably resulted from the inhibition of transcription.

To better measure theeffectofcoumermycin on phage RNA and protein synthesis, we also carriedoutthis

experiment

usingcells in which hostsynthesiswasshut off

prior

tophage infec-tionby irradiation with UVlight (Fig. 2d,e,and

f).

It is apparent that phage transcription is

significantly

inhibited by the

antibiotic,

espe-ciallyatlatertimesduringinfection.Thus,at6,

s0

a,

N 60

m

20

0

1 25 5 10 20 50 100

ug/ml COUMERMYCIN

FIG. 1. Coumermycin inhibits the growth of bac-teriophage T7. One-step growth experiments were

carriedoutin Tbrothat30°Casdescribed by Baird

etal. (1).At 5min after infection, infectivecenters

weredilutedintobroth containing the indicated final concentrations of coumermycin. The burst size is presentedasthepercentageofacontrolcontaining no coumermycin. The bacterial strains used were:

B/l (A);NI747(0);NI748Cou' (0).

9, 12, and 15 min after infection coumermycin

inhibited RNAsynthesisby47, 58,63,and 81%, respectively.

Todemonstrate that the effect of coumermy-cin on both T7 DNA and RNAsynthesis results from inhibition of the E. coli DNA gyrase, we determinedthe effect of the antibiotic on phage DNA, RNA, and protein synthesis using the isogenic K-12 strains NI 747 Cous and NI 748 Cour (Fig. 3).The addition of30,ug of coumer-mycin per ml to the resistant strain had little affect onT7 DNA, RNA, or protein synthesis (Fig. 3a, b, and c), whereas the coumermycin-sensitiveK-12strain gave resultssimilartothose found for E. coli B/1 (Fig. 3e, f, and g). The experiments shown in Fig. 3 were done using UV-irradiated cells, but similar results have beenobtainedusing non-irradiated cells.

The effectsofcoumermycinconcentration on the inhibition of T7 DNA, RNA, and protein synthesisarecompared in Fig. 4. The response tocoumermycinwassimilar for the inhibition of bothDNA andRNAsynthesis, althoughinboth casessynthesiswas more sensitive to the anti-biotic at later timesduringthe infection.

Insummary, coumermycin causes an almost complete inhibition of phage DNAreplication andsignificantly reduces the rate of phage tran-scription.Thisstronglysuggeststhat the E. coli DNA gyraseplaysanimportantrolein both T7 DNA andRNA synthesis.However, it is possible thatDNA gyraseisnotrequiredfor phage nu-cleicacid synthesisbut that,inthe presence of coumermycin, a drug-enzyme complex acts as

an inhibitor. Although this second possibility

seems

unlikely,

it cannot be ruled out at the

presenttime.

Transcription of early genes is not in-hibited by coumermycin. The results pre-sented above indicate thatatearly times during infection T7 transcription is only slightly in-hibited by coumermycin. To test directly whetherearly transcription is inhibitedby the antibiotic, we measured the effect of coumer-mycinon transcription duringinfection witha T7gene1amber mutant. The T7 gene 1 protein isanRNApolymerase that is required for tran-scription of the class II and class III genes. During infection by gene 1 mutants, only the class I or early genes are transcribed. In the absence of the gene 1 RNA polymerase, T7

transcriptionwascompletelyunaffectedby

cou-mermycin (Fig. 5a). This suggests that DNA

gyrase is not required for transcription of the early genes by thehostRNApolymerase.

Coumermycininhibition of T7 transcrip-tion doesnotrequireDNAsynthesis.Since

DNAgyraseplaysan essential role inT7 DNA

0

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10

20

I0

U

i

0

7 20

-4-~~~ ~ ~ ~ ~ ~

~~~i-120--~~~~~~

4-i~~~~~~~~

0 10 2 0 1 0 10 0 30 10 I 10

20i 30i

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MI NUTES

FIG. 2. Effect of coumermycinonT7DNA,RNA,andproteinsynthesisin normal andLW-irradiatedcells.

TherateofDNA (a, d),RNA(b,e),andprotein (c,f) synesiswasmeaswuredatintervalsafterinfection of

normal(a,b, c)orUV-irradiated(d,e,f)E. coliB/1withT7phageasdescribedin thetext. Threeminutes

before infection, 30pg of coumermycinperml (0) oranequivalent amountofthe drug diluent dimethyl

sulfoxide(0)wasaddedtothe culture.

U

a.

CL)

6

i

2

0

6

i

2

0

r--s_-

104-20ti----30 A1O0 04-+10 720i-4--4-30 100 0 -1-MINUTES

FIG. 3. Effect ofcoumermycin on T7DNA, RNA, andprotein synthesis incoumermycin-sensitive and -resistantcells.Procedureswereas described in thelegendtoFig.2.E.coli NI 748Cou' (a, b, c) and NI 747 Cou'(d,e,g)wereirradiated with LWlightpriortoinfection.Control(0);30pgofcoumermycinper ml(0).

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~~~~~20j

3010

0 10 20 30 *0 0 10 20 30 10 0 10 20 30 10

MINUTES

FIG. 4. Effectof coumermycin concentrationonT7 DNA, RNA, and protein synthesis.Procedureswere asdescribedinthelegendtoFig.2. E.coliB/l was

irradiated with UVlight,and theindicatedamounts of coumermycinwereadded 3minpriortoinfection withT7phage.Control(0); coumermycinat3,g/ml (0); 10 pg/ml (A);30pg/ml(0); 1)00

jg/ml

(U).

replication,itseemed possible that theinhibition

of late transcription bycoumermycin might be

asecondary effect resulting from the inhibition

of DNAsynthesis. Late transcription of T7 does

notnormally require DNA replication, but

abor-tive replication in the absence of DNA gyrase

might produce lesions in the DNA which inhibit

transcription. Itwastherefore of interest to

de-termine whether DNAsynthesis is required for

coumermycin inhibition of transcription. T7

pro-videsanexcellentsystemto testtherequirement

for DNA synthesis, since a number of phage

mutants are defective in DNA replication but

notintranscription. We measured the effect of

coumermycinonphagetranscription incells

in-fected with T7 gene 5 (DNA polymerase) and

gene 6 (exonuclease) mutants. The T7 DNA polymerase is required for all stages of phage

DNA replication. However, during infection

withaT7gene5mutant,theinhibitionof

tran-scription by coumermycinwas identicaltothat

observed duringawild-type infection (Fig. 5b).

The T7 exonuclease is not required for early

stages ofphage DNAreplication, but is

appar-ently required for the formation oflarge

repli-cativeintermnediates. As wil be discussedlater,

structures containing up to several hundred

phage equivalents of DNA (concatemers) are

formedduringreplication in normal infections.

Concatemersarenotfoundincellsinfectedwith

T7 gene 6 mutants (12). However, the gene 6 mutant infection exhibited a normal

coumer-mycin inhibition ofbothtranscription (Fig. 5c)

and DNA synthesis (data not shown). Thus,

coumermycin inhibition of transcription does

notrequireDNAsynthesis,whichsuggeststhat

DNA gyrase plays a directrole inphage RNA

synthesis. These data also indicate that DNA

gyraseisrequiredfor DNAand RNAsynthesis

even when large replicative intermediates are

not formed and when the template DNA

re-mains inunit-length linear molecules.

Coumermycin selectively inhibits the synthesis of late proteins. Since our studies

on the kinetics of phage RNA synthesis

sug-gested that late transcription is preferentially inhibited by coumermycin, itwasof interestto

determine the effect of the antibiotic on the patternsofprotein synthesisduringphage infec-tion. Although protein synthesis is not as

strongly inhibited by coumermycin as is DNA

or RNA synthesis, it seemed possiblethat the pattern ofprotein synthesismight be altered by the drug. For example, the synthesis of func-tionallate mRNAmightbecompletelyblocked by theantibiotic,andprotein synthesisobserved

at late times in the presence of coumermycin could represent residual translation of early mRNA.Alternatively,thetranscription ofsome

lategenescould beaffectedmorestrongly than

transcription of others. To examine these possi-bilities, we labeled T7 proteins with

"4C-amino

acidsatintervalsduring infection in thepresence

andabsence ofcoumermycin.Theproteinswere

then analyzed by electrophoresis in polyacryl-amide gels containing sodium dodecyl sulfate (Fig. 6).

Synthesis of T7 early proteins, the products

of genes which are transcribed by the E. coli

RNApolymeraseandareexpressedimmediately after infection (e.g., genes 0.7, 1, and 1.3), was notaffectedby coumermycin. Thissupports our

previous conclusion that transcription of the

earlygenesisinsensitivetothedrug. Incontrast,

synthesis of many ofthe late proteinswas

se-verely inhibited by coumermycin. However,not

all of the genes transcribed by the T7 RNA polymerase were affected equally by the anti-biotic. Theexpressionof theclassII genes 4, 5,

6, and HDP (helix destabilizing protein)

ap-peared to be relatively insensitive to

coumer-6 10- 10 -

-0 10 20 30 10 0 10 20 30 10 0 10 20 30 10

MINUTES

FIG. 5. Effect of coumermycin on RNA synthesis in cells infected with T7 gene 1, gene 5, or gene 6 amber mutants. E. coliB/i was irradiated with UV lightand infected with T7phage bearing an amber mutant in gene1 (a), gene 5 (b), or gene 6 (c), and the rate of RNA synthesis was measured at intervals afterinfection as described in the text. Control (0); 30 pgof coumermycin per ml (0).

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COUMERMYCIN INHIBITS T7 DNA AND RNA SYNTHESIS 533

2

4

6

8

10

12

14

16

18

_ + + 1- 4- z _ .i _44+ _ +

"Wo -16

. x

4b~~~ 41U1 ~~4qu -1WW

tsp~~~~~~~~~~lY

_;

-12

ftair~~~~~~~~~~~~~~~~.*_W -_-i.LI9;;

_P

S* ,S,

,.W

10

-10

-6

HDP

-FIG. 6. Polyacrylamide gel electrophoresis ofT7proteins synthesized in the presence or absence of

coumermycin. Procedureswereas describedby Studier(20,21). UV-irradiated E. coliB/l wasinfected with

T7phageatamultiplicity of 10 in M9 medium at30°C. At 2-min intervals 0.1-mi samples of the infected culture wereaddedto10 ,Llofamixturecontaining"4C-aminoacids (200X MCi/ml) and 1mgof unlabeled

arginineper ml.After 2 minat30°C,1mlof T brothwasaddedtoeachsample, and incubation continued for 4min. The cells were then collectedby centrifugation, suspended in 50 mM Tris (pH 6.8) containing 1%

mercaptoethanol, 1%sodiumdodecyl sulfate,and10%glycerol,incubatedfor2 minat100°C,andsubjected

toelectrophoresisinadiscontinuous sodiumdodecylsulfate-containing buffer systemon a10%polyacrylamide

slabgeL Radioactivityonthe driedgelwasvisualizedby autoradiography. The number at thetopofeach

track is the time(minutes) after infectionatwhich thepulseof '4C-aminoacidsbegan forthe control culture (-) andaculturecontaining50pgof coumermycinperml(+). The numberoftheT7genethatspecifieseach proteinasidentified by Studier (20; personalcommunication) is givenatthe sides. The helix-destabilizing protein (HDP) hasnotbeenassignedagenenumber.

mycin, whereastheexpressionofgenesnearthe

rightend of the T7genome (for example,genes

12, 15, 16, 17, and 19)wasseverelyinhibited.

Thus, T7geneexpressionexhibitsagradient

ofsensitivitytothe antibioticcoumermycin.The

expression of earlygenes,at theextreme left of

the T7genome,is insensitivetothedrug,while

the expressionofgenesat the right end of the

genome isseverelyinhibited. Theexpressionof

genes in the middle of the genomeexhibits

in-termediatelevels of inhibition.

DISCUSSION

E. coli DNA gyrase is required in vivo to

maintain acircular DNA inanunderwound or

negatively supercoiledstate.Thishas been

dem-onstratedforsuperinfecting phagelambda DNA

(5) and more recently for the folded bacterial

chromosome itself (3). Itseems likelythat the

negative supercoiling of a circular DNA may

provide an important driving force for DNA

replication, transcription,and recombination. It

has been shownthat in vitro such DNA

mole-culescanhybridizewithsingle-strandDNA

frag-ments to form the triple-stranded joint

com-monly postulatedas anintermediate in

recom-bination (7). Furthermore, site-specific

recom-bination ofphage lambda in vitro requires

su-perhelical DNA (13). Infact, DNA gyrasewas

first identified on the basis of its role in this

reaction (14). The transcriptionofanumber of

circular DNAsin vitro(2,16,24)has been shown

to be enhancedby superhelicity. Inreplication,

maintining the DNA in an underwound state

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would aid the necessaryunwindingatthe grow-ing fork. Inaddition, in the replication of 4X174 RFDNA,superhelicity has been shownto pro-motethe binding of the phage gene A protein, which isrequired to initiatereplication (10).

Thus DNA gyrase may wellplayanessential role in thereplication, transcription, and recom-bination of circular DNAs. Indeed, coumermy-cin,which inhibits DNA gyrase in vitro (5),also inhibits bacterial DNA replication and to a

lesser extenttranscriptionin vivo(17). However, it isless clear how DNA gyrase functions in T7 DNAreplicationandtranscription. The T7 ge-nome is a linear duplex DNA molecule, and duringatleast the first round ofreplicationthe DNA appears to remain in a linear structure

(25), with no obvious topological constraints. During subsequent rounds of replication the DNAisfound inlinear concatemers,containing up to 10phageequivalents of DNA, and then in much larger structures containing several hundredphage equivalents of DNA (4, 9, 15, 18, 19). Theselarge loopedstructuresappear similar

to the folded bacterial chromosome and the unwinding of this DNA may wellrequire topo-isomerasessuch asX (23) and DNA gyrase (5). Unwindingof this DNA doesnot appearto be constrained invitro,since the addition of ethid-ium bromide doesnotproduce supercoiledDNA (15).However,it seemsquitelikelythat in vivo theselarge replication structurescontain topo-logicallyisolated DNAloopsanalogoustothose found in the bacterial chromosome (25). Thus,

atleastduringthe later stages ofphagegrowth

itseemsreasonable thatphageDNAreplication

andtranscriptionwould be affectedbythe DNA gyrase inhibitor coumermycin. In fact, wefind thatphagenucleic acidsynthesisis less sensitive

tocoumermycin atearlytimes duringinfection than it isatlatertimeswhen thelarge replicative intermediates are formed. This is particularly

true of RNA synthesis, where only late tran-scriptionappearstobe inhibitedby coumermy-cin; but with DNA synthesis, which is more

completelyinhibited bythe antibiotic, wealso

see a smallamount ofsynthesis atearly times

(10 to 15min) even atvery highcoumermycin

concentrations (for example, Fig. 4). This sug-geststhat asmallamountofphageDNA repli-cationcan occur inthe absence of DNAgyrase activity. However, the formation oflarge

con-catenated DNAintermediates isapparentlynot

necessaryfor coumermycin inhibition of DNA

and RNAsynthesis. We foundanormal

inhibi-tion of both DNA synthesis (data not shown) and RNAsynthesis(Fig.5)duringinfectionwith

a T7 gene 6 amber mutant, although

concate-mers areapparentlynotformed duringthis

in-fection (12). Inaddition,a normalinhibition of

RNA synthesis was observed during infection with a T7 gene 5 mutant, where no DNA repli-cation is observed. Therefore DNA gyrase ap-pears tobe required for efficient phage nucleic acid synthesis even under conditionswhere large replicativeintermediates are not formed.

Consistent withthis, Itoh andTomizawa have shown thatcoumermycinblocks even the first round of T7 DNAreplication (8).During infec-tion in the presence of a density label, they found thatvery little of theinfecting DNA was converted to ahybriddensity in the presence of coumermycin, suggesting that DNA gyrase is required for efficient completion of the first round of T7 DNAreplication.

Since DNA gyrase is required for T7 DNA synthesis invivo, it is surprising that the enzyme is notrequired for DNAsynthesis in vitro. We have previouslyshown thatnalidixic acid, now known to be an inhibitor of DNA gyrase (22), hasessentiallynoeffect on the rate of T7 DNA synthesis in a cell-free system (6). More recently wehave also shown that coumermycin, at con-centrationsupto100,ug/ml, has no effect on T7 DNAsynthesis in vitro (D. Hinkle, unpublished data). Yet this cell-free system supports exten-sive DNAsynthesisand produces abiologically active product (11). Perhaps attachment of the DNA tothe cell membrane and/or polysomes in vivo produces topological constraints that are notpresent invitro. This may also explain why DNA gyrase is requiredforefficient transcrip-tion of thelate,butnottheearly, T7 genes, even in the absence of DNA synthesis. Immediately after infectionthe DNA is presumably relatively free of polyribosomes and other constraints, so that the limited unwinding of the DNA helix, which takes place during the binding of RNA polymerasetothe promoter and thesubsequent RNA synthesis, can occur in the absence of DNAgyraseactivity. As the infection proceeds, the constraints onthe unwinding ofthe DNA helixmay become moresevere, resulting in the observedgradient of coumermycin inhibition of T7proteinsynthesis (Fig. 6).Alternatively, the

T7 RNA polymerase may bind to and unwind

some T7promoters moreeasilythanothers,so

thatthe negative torque provided by DNA gy-rase stimulatesthe transcription of some genes more than others.

It will be interesting to determine whether DNAgyrase affectsthepatternoftranscription by the purifiedT7RNApolymerase.Inaddition,

as systems are developed forreplication of T7 DNAusingpurifiedproteins,the effect ofDNA

gyraseonreplicationcan be testeddirectly.

ACKNOWLEDGMENTS

WearegratefultoKarl Drlica formanyhelpfuldiscussions J. VIROL.

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INHIBITS T7 DNA AND RNA SYNTHESIS 535

andtoR.BruceStruthersforassistance with the computer graphics.

Thisinvestigationwassupported by Public Health Service grantGM-21782 from theNational Institute ofGeneral Med-icalSciences andby grant NP 179 from the American Cancer Society, Inc. M.D.W. issupportedby Public Health Service training grantGM-07102,and D.C.H. is arecipientof Public Health Service CareerProgram Award GM-00221, both from the National Institute of General Medical Sciences.

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3. Drlica, K., andM. Snyder. 1978. Superhelical Esche-richia coli DNA:Relaxationby coumermycin. J.Mol. Biol. 120:145-154.

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5. Gellert, M.,M. H.O'Dea,T.Itoh,andJ.-I. Tomizawa. 1976. Novobiocinandcoumermycin inhibit DNA su-percoiling catalyzed byDNA gyrase. Proc.Natl. Acad. Sci.U.S.A.73:4474-4478.

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10. Marians, K.J., J.-E. Ikeda, S. Schlagman, and J. Hurwitz.1977.Role of DNAgyraseinOX replicative-formreplicationin vitro. Proc. Natl. Acad.Sci.U.S.A. 74:1965-1968.

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12.Miller, R. C., Jr., M. Lee, D. G. Scraba, andV. Paet-kau.1976.TheroleofbacteriophageT7endonuclease (gene 6) in geneticrecombination and production of concatemers. J. Mol. Biol. 101:223-234.

13.Mizuuchi, K., M. Gellert, and H. A. Nash. 1978. In-volvement of supertwisted DNA in integrative recom-bination ofbacteriophage lambda. J.Mol. Biol. 121: 375-392.

14.Nash, H. A. 1975. Integrative recombination of bacterio-phage lambda DNA in vitro. Proc. Natl. Acad. Sci. U.S.A.72:1072-1076.

15. Paetkau, V., L.Langman,R. Bradley, D.Scraba, and R.C. Miller, Jr.1977.Folded,concatenated genomes asreplication intermediatesof bacteriophage T7 DNA. J.Virol. 22:130-141.

16. Richardson, J. P. 1975. Initiationoftranscription by EscherichiacoliRNApolymerasefromsupercoiledand non-supercoiled bacteriophagePM2 DNA. J. Mol. Biol. 91:477-487.

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