Inhibition of bacteriophage M13 and phix174 duplex DNA replication and single-strand synthesis in temperature-sensitive dnaZ mutants of Escherichia coli.

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Copyright 0 1977 AmericanSociety forMicrobiology PrintedinU.S.A.

Inhibition of Bacteriophage M13 and

4X174 Duplex DNA

Replication and Single-Strand Synthesis

in

Temperature-Sensitive

dnaZ

Mutants of

Escherichia coli

WILLIAM G. HALDENWANG AND JAMES R. WALKER* Microbiology Department, University of Texas, Austin, Texas 78712

Received forpublication8October1976

AfunctionaldnaZ product, known to be essentialfor host DNA

polymeriza-tion and for the synthesis of M13 and

OX174

parental replicative-form (RF)

DNA, isrequired also for RF replication and single-strand synthesis bybothof

these phages. Allthree stages of M13and4X174DNAreplication (parentalRF

formation, RFreplication, andsingle-strand synthesis) are inhibited in dnaZts

mutants at elevated temperatures. Inaddition, the thermolabile step in M13

parentalRFformationappears to occurafter RNApriming; i.e.,thesynthesisof

M13 RF DNA proceeded when adnaZts mutant, infected at anonpermissive temperature, was transferred to a permissive temperature in the presence of rifampin.

Thesingle-strand (SS) DNA phages M13 and

4X174requirehostDNAreplicativefactors for

the synthesis oftheir replicative intermediate

and progeny viral DNAs (13, 17). Several Esch-erichia coli mutants, withdefectsinhost chro-mosomereplication,aredeficientingene prod-uctsthat arerequiredby these phages in one or more stagesof theirDNAreplication cycle: pa-rentalreplicative-form (RF) synthesis, RF

rep-lication, or the asymmetric synthesis of

prog-enyviral strands (forasummary, see 7).

We havepreviously presentedevidence that

theproduct of the DNAsynthesis gene,dnaZ,

isneededinvivo for the first stage of M13 and

OX174

replication, the conversionofinfecting viral SS DNA into duplex parental RF DNA (10); Wickner and Hurwitz (21) reported that the dnaZ gene product is required in vitro for DNA polymerase mI activity on primed SS M13,

4bX174,

andST-1templates. The observa-tionthat the dnaZfunctionisrequired for DNA

polymerase III-catalyzed DNAsynthesis in

vi-trosuggeststhat the dnaZproductmay partici-pate in vivo in all stages of M13 and

OX174

replicationthat have been shown to beeffected

byDNApolymerase III,i.e., RFreplicationand

SSsynthesis (3, 8, 18) (in additiontoparental

RFformation).

In this communication, we report that the replication of M13 and

4X174

RF DNA and the synthesis of progeny SS DNA are inhibitedin dnaZts mutants at nonpermissive

tempera-tures. In addition, the dnaZ function in M13

parentalRFformationinvivoappears tooccur

at a step after RNA priming. These data

sug-gest a function for the dnaZ protein in the elongation phase ofinvivoDNAsynthesisorin

aninitiationstepthat occursafterthepriming

reaction.

MATERIALS AND METHODS

Bacterial and phage strains. K-12 strain AX727

dnaZts2016 and the 4X174-sensitive K-12 strain

GN727dnaZts2016 were describedpreviously (10). H727 is adnaZts2016recombinant between H5274

(obtainedfrom D. Ray) andHAX727(Hfrof AX727

constructedby F'lac+ integration)and has the geno-type K-12 F- dnaZts2016 thy rpsE tsx. C727 is a

dnaZts2016 recombinant between E. coli C

(ob-tained from R. McKee) and HAX727. It has the

genotypeC F-dnaZts2016rpsEtsx. F'101/C600was obtained from B. Bachmann, the male specific phage M13 from D. Ray,4)X174h8p(retarded lysis)

from C. Earhart, and 4X174am3 (lysis defective)

from R. L. Sinsheimer. AX727 TS+, GN727 TS+,

H727TS+,andC727TS+werespontaneous

tempera-ture-insensitiverevertantsofAX727, GN727, H727,

and C727. F'lac+ derivativeswereobtainedby mat-ing withF'lac/RV (obtainedfrom M. Malamy).

Media. Yeast extract-tryptone (YET) broth has

been described (20). YET brothwas supplemented

with [3H]thymidine at 10 ,uCi/0.12 ,tg per ml for

radioactive labeling. Minimal phosphate medium

was 0.8% nutrientbroth (Difco), 1.5 mM NH40H,

and2.5mMMgSO4(pH 7.0).

Preparation ofphagestocks and marker DNAs. Nonradioactive M13 was prepared by infecting F'101/C600 (4 x 108cells/ml)inYETbroth with M13

at a multiplicityofinfection of50. Aftera 30-min

adsorption periodat30°C, the culture wasdiluted

1:50and growninYET broth at30°C to acelldensity of7 x 108cells/ml. Cellswereremovedbylow-speed

centrifugation, and the phagewasconcentratedby

23

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24 HALDENWANG AND WALKER pelleting as described previously (10). 32P-labeled M13 wasgrown similarly in minimalphosphate me-dium supplemented with 20

tiCi

of carrier free

32p043- per ml. After concentration as above, the

phages were purified on high-salt, 5 to 20% neutral sucrose gradients (10) and the viral DNA was iso-lated by cold phenol extraction (2).

OX174am3

and bX174h8pweregrown in E. coli C inYET brothsupplemented with 1.5 mg ofCaCl2per ml. E. coli C cultures (2x 108 cells/ml) were infected with either4X174am3or

OX174h8p

at amultiplicity ofinfection of 3 and grown for 2.5 h at 370C. The cultures were concentrated 25-fold into 0.1 M Tris-hydrochloride (pH 8.1) containing 0.5 mg of lyso-zyme per ml and 10 mM EDTA. After a 60-min incubation on ice, lysis was completed by shaking with a fewdrops of chloroform. To decrease viscosity and separate phage from debris, the lysate was drawn through a 0.5-inch (about 1.3-cm), 26-gauge needle. Debris wasremoved by low-speed centrifu-gation. Phage that were to be used as a source of marker DNA (4X174h8p) were gradient purified, and the DNA was extracted by the method of Brown and Dowell (2).

Analysis of M13 and

OX174

DNAsynthesis. Ter-mination of isotopeincorporation and lysis of M13-infected cells were accomplished as described by Ray etal. (15). 4X174am3-infectedcells were processed by the protocol of Dumas et al. (4), except that the lysate was incubated with Sarkosyl for 10 min at

370Cbefore Pronase treatment, rather than for 16 h at 0°C. Crude lysates of M13- and

OX174am3-in-fected cells were analyzed on high-salt, 5 to 20% sucrose gradients (10). 32P-labeled M13 DNA and unlabeled 4X174h8pDNA wereapplied to the gra-dients as SS markers for M13- and

OX174am3-in-fected cell lysates, respectively. The position of the

OX174marker wasassessed by itsbiologicalactivity

in the spheroplast infectivity assay (9). Gradient fractions were collected from the bottom. Samples of 100

Al

were spotted onto GF/A glass fiber filters,

which, after drying, were washed with 3 ml of 5%

trichloroacetic acid, dried, and counted.

RESULTS

Role of dnaZ in M13 parental RF forma-tion. An active dnaZproductisrequired for the synthesis of both M13 and

OX174

parental RF

DNA(10). InasmuchasdifferentRNA priming

mechanisms are used by each of these phages

(16), itmight be assumed that the dnaZ

prod-uctwouldnotbe involvedinthepriming step of RFsynthesis. This assumption could be tested

inthe M13system. RNA priming on M13

infect-ingSSDNA isbelievedto beeffectedby

rifam-pin-sensitive RNApolymerase (16). Therefore,

ifinfectionofadnaZts mutant by M13 at a

non-permissivetemperature rendered parental RF formationresistant torifampin when the

tem-perature subsequentlywaslowered, itmaybe

concluded thattherifampin-sensitivestep, i.e.,

priming,could be accomplishedintheabsence

ofanactivednaZproduct. Itwaspossibletodo this experiment because the product of the dnaZts2Ol6 allele regains activityimmediately upon temperature reduction even in the

ab-senceofproteinsynthesis (Chuetal.,in

prepa-ration). A culture of F'lac+/AX727 was grown at

300C

and divided into threesubcultures, all of whichwerethenincubated at410Cto inacti-vatethe dnaZproduct. Rifampinwasadded to

twoofthese cultures either 5 min before or 5 minafterinfection by M13. AsdescribedinFig. 1, the cultures were then transferred to 30°C

and pulse-labeled with [3H]thymidine. The

third culture wasinfected andpulse-labeledat

41°C to demonstrate that no RF synthesis

oc-curredatthistemperature.

InfectionofadnaZtsculturebyM13at410C resultedin asubstantialamountof RF

synthe-sis upon return ofthe culture to 30°C in the

presenceof rifampin(Fig. 1A).Culturestreated with rifampin before infection yielded no de-tectable peaks ofphage DNA (Fig. 1B). There

was no apparent RF synthesis in the culture

infected andpulse-labeledat41°C(Fig. 1C). We

infer that RNAprimingofinfectingM13

single

strands canoccurinvivo in the absence ofan

activednaZ product.The culture infected with

M13 before rifampin treatment (Fig. 1A)

con-tainedtwopeakswithsedimentationproperties

consistentwith RF IandRF II. The

magnitude

of the RFIIpeak(fraction27)was

unexpected.

Formationof RF II DNAnormallyrequires the

gene 2protein of M13 (5), andthesynthesis of

this protein should have been inhibited in a

rifampin-treated culture. The amount of

la-beled material thatsedimentedasRF IIvaried fromexperimentto experiment; this variation might have been aresult of the length ofthe

410C

preincubation period rather than an

in-complete rifampinblockonM13gene 2protein

synthesis. Chloramphenicol, atconcentrations

thatareadequatetopreventRF IIformationin

M13-infected cells(10), failedtoeliminate RF II material fromlysatesofF'lac+/AX727 cultures

thatwerepreincubatedat

410C

andthen

trans-ferred to 30°C (data not shown). It is possible

that thematerialformingthe RF IIpeak

repre-sented abnormalDNAsynthesizedinthe

pres-enceofapartiallyreactivated dnaZproductor,

alternatively, RFIbreakdownproductsformed

as aresult ofanucleolytic activitypresentafter the 41°C incubation.

Requirement forthednaZ function in M13

and 4X174 growth afterparental RF

forma-tion. The effect of the dnaZ mutation ofM13

and

4X174

phage growth after parental RF formation was tested by shifting dnaZts and

TS+ cultures, which were infected and

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M13 AND

OX174

DNA SYNTHESIS IN dnaZts HOSTS M13 RIF 3H 30C

5" 4 1I 10 _41*C _3H_M13

C

₃₀₀i I"₁ 1_IS"

Io

FRACTION NUMBER

FIG. 1. Formation of M13 parental RFat410C byadnaZts culture in thepresence of rifampin. F'lac+I

AX727dnaZtswasgrowntoadensity of3.5 x108cells/ml in YET brothat30°C. Three sampleswerethen

transferredto41°C. Culture A (0)wasincubated for 30 minat41°C andinfected with M13 (multiplicity of infection =100).Five minutesafter infection, rifampinwasaddedtoaconcentrationof200 pg/mland the

incubation of 41°C was continued for an additional 5 min. The culture was thenpulse-labeled with

[3H]thymidine for 10 minat30°C. Culture B (@)wassimilarly treated;however, rifampinwasadded5min

before infection by M13.Culture C(U)wasnotexposedtorifampin butwasinfected and pulse-labeledat41°C. Cellswereharvested, lysed, and sedimented through high-salt neutralsucrosegradients. Sedimentation in

this andall othergraphs wasfrom righttoleft. The verticalarrowindicates the sedimentation position of

P2P]labeledM13 SS DNA.

bated at a permissive temperature (to allow

parental RF formation), to either 41 or 40°C

(nonpermissive for DNA synthesis).

Cultures of F'lac+/H727 dnaZts and TS+

were grown and infected with M13 at 300C.

After 30min,half of each culturewasshiftedto

41°C. Although phageproduction in the dnaZts

and TS+ cultures was similar at 30°C, the

dnaZts culture terminated phage synthesis

after 30min at410C (Fig. 2A). M13 growthwas

slowed in the revertant culture at 410C;

how-ever, phage production continued throughout

the course of the experiment. (The previously

reported[201 immediate cessation of M13 phage

production in another dnaZts strain [AX727]

appears to result from an immediate halt of

M13 SS synthesis, by temperature elevation,

whichprobably is unrelatedtothe dnaZ

muta-tion. M13 SS synthesis alsowas inhibited,

al-though transiently, after a shift ofan AX727

TS+ cultureto 41°C [unpublished data].)

Shifting C727 dnaZts and TS+ cultures to

400C after they were infected with qbX174am3

andincubated at350C (selectedasthe

permis-sive temperature) for 15 min inhibited phage

growth only in the dnaZts culture (Fig. 2B).

Although phage productioninthe dnaZts

cul-ture wasdetectable at 400C foras longas 105

min, the rate ofphage production, relativeto

that in therevertantculture, appearedto slow

after 15min atthis temperature. Total phage

yield in the dnaZts culture after 165 min at

400C was approximately 1/100 the yieldinthe

revertant culture. Phage growth in these cul-tures was comparable at 350C. Inhibition of

4X174growthwaspreviouslyobservedby

Tak-eto (19) in AX727 (dnaZts) spheroplasts that

weretransformed with 4X174 RFDNA.

41*C

30a

41C

RIF M13 3H 30C

25 5 4 1"

A

B

16

cm 12

1 0

X 8

04 0 4

25

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26 HALDENWANG AND WALKER

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I-MINUTES

FIG. 2. Growth of M13 and 4X174am3 in dnaZtsand TS+ cultures. (A) M13. Culturesof F'lac+/H727

dnaZts and TS+were grown at300CinYETbrothsupplemented with2 pgofthymine per ml to a densityof2

X108cellslmlandconcentrated to 2 x 109 cells/ml in buffer (0.01 Mpotassium phosphate[pH7.0] and 0.01 M

MgSO4).M13 was added(multiplicityof infection =50),and the suspension was incubated for 30 min at

300C. Thecells were then washed twice inbufferand diluted to aconcentrationof 2 x 103cells/mlin YET broth (plus 2 pgof thymine per ml) at300C. After30 min at thistemperature,halfofeach culture wasshifted

to41°C.Samples were taken at the times indicated andimmediatelyplatedonF'101IC600.dnaZts cultureat 30°C (A) and at 41 °C(A); TS+ culture at 30°C (0) and at 410C (@). (B)OX174. Culturesof C727 dnaZts and TS+ were grown at 30°C in YET brothsupplementedwith 1.5 mgof CaCl2 per ml to a celldensity of2 x108

cells/ml, concentrated10-fold into 0.01 MMgSO4and 0.01 MCaC12, andinfectedwith

OX1

74am3

(multiplic-ity ofinfection =5)for 15 min at30°C. The cells were thenwashedtwice inthe same solution and diluted to2 x 107cellslml in YET broth(containing 1.5 mg ofCaCl2per ml) at35°C. After 15min, halfof each culture wasshiftedto40°C.Samples of0.1 ml weretransferredto1.9 mloflysis buffer(0.1 M Tris[pH8.11,0.5 mgof

lysozymeperml, and 10mMEDTA) at the indicated times.Samplesinlysis bufferwereincubatedfor60 min

onice, vortexed with adropofchloroform,and titered on G727 TS+ (a host that can suppress the

OX1

74am3 mutation). Symbols are as in (A).

We conclude that the dnaZ function is

re-quired by both M13 and

OX174

for continual

phageproduction after the formation of

paren-tal RF DNA. It is unclear whether the

differ-ences in the rate of termination of M13 and

X174am3 phage growth at nonpermissive

temperatures represent differentrequirements

for the dnaZ product. Given therapid rate of

4PX174

growth relativetothatofM13, however, prolonged

OX174

phage production at 400C

might reflect packaging of previously

synthe-sized 4X174viral DNAorpossibly slower

inac-tivation of the dnaZ product at 400C than at

410C.

Requirement for the dnaZ function inM13

and

OX174

RF replication. To determine

which stages ofphage DNA replication, after

parental RF formation, were inhibited in the

dnaZts mutants, we firstexamined RF -- RF

replication. F'lac+ derivatives ofdnaZts and

TS+ cultures were infected at 350C with M13

and heldatthistemperaturefor 10min.

Pulse-labelingduring this perioddemonstrated (Fig.

3A) the formation of RF DNAinboth the mu-tant and revertant cultures atthis permissive temperature. Duplicate cultures thatwere sim-ilarly infected andheldat

350C

for10 minwere then incubated for an additional 15 min at either 35 or

410C.

Pulse-labeling the cultures

that wereheld at

350C

revealed (Fig. 3B) RF

and SS DNAsynthesisinbothtsand TS+

cul-tures;however, incubation at41°C terminated

M13-specific DNA replication in the dnaZts

mutant(Fig.

30).

The asymmetric synthesis of M13 SS DNA beginsatapproximately10minafter infection (13). Therefore, itispossible thatasignificant

amount ofRF DNA present in Fig. 3B and C

represented intermediatesinSSsynthesis. The inhibition ofDNAsynthesisinthe ts culture at

410C

may thusreflectadnaZproduct

require-ment in asymmetric synthesis and not RF -*

RFreplication. This ambiguitywasresolved by

treatingM13-infected dnaZts and TS+ cultures

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4X174 dnaZts

1.5 I.0 0.5

I.

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50

5

10

8 16 24 32 40 48

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FRACTION NUMBER

FIG. 3. Replication of M13 RF DNA in dnaZts andTS+ cultures. F'lac+/H727 dnaZts and TS+

cul-turesweregrownto3.5 x108cellslml inYET broth

supplementedwith 2pgof thymidine, transferredto

35°C,andinfectedwithM13(multiplicity of infection

=100). The verticalarrowindicates thepositionof

32P-labeled M13 SS DNA marker. (A) dnaZts (@) andTS+ (0) cultures pulse-labeled for 10min after infection at35°C. (B) dnaZts (@)andTS+ (0)

cul-tures, infectedasabove, were incubatedat35°C for 25 minafter the addition of M13 andpulse-labeled for an additional 5 min with [3H]thymidine. (C)

dnaZts (@) and TS'(0)culturesweretransferredto

410Caftera10-min infection periodat35°C. Fifteen minutesafterthetemperatureshift, thecultureswere

pulse-labeled foranadditional 5 min.

with chloramphenicol before pulse-labeling.

Chloramphenicol, atconcentrations of10 to 30

,ug/ml, selectively inhibits M13 SS DNA

syn-thesis while permiting the replication of M13

RF DNA (14). Cultures of F'lac+ derivatives of

dnaZtsand TS+ strainswereinfected with M13

at 350C and, after 10 min, a portion of each

culturewasshiftedto400C.Afteranadditional

10min (20 min postinfection), chloramphenicol

(30 ,g/ml) was added to each of the cultures

and the incubation was continued for 10 min.

Pulse-labeling after chloramphenicol

treat-mentdemonstrated the synthesis of RF DNA in

dnaZts and TS+ culturesat35°C (Fig. 4A) and

in the TS+ culture at 40°C (Fig. 4B) but

vir-tuallyno M13 RFsynthesis in the dnaZts

cul-ture at400C (Fig. 4B).

OX174am3-infected

cells. Chloramphenicol, at aconcentration of 30

,g/ml,

permits the forma-tion and replication of

OX174

RF DNA while inhibiting SS DNA synthesis (11). Chloram-phenicol-treated (30 ug/ml) dnaZts and TS+ cultures of E. coli C were infected with

OX174am3

at 350C. A portion of each culture wasshiftedto400C after5min. Duringthis 5-mininfection intervalat350C,RF Iand IIwere formed (datanotshown). After a20-min incu-bation periodat 35 or400C (25 min post-infec-tion), the cultureswerepulse-labeled for5min with[3H]thymidine. Incubation of the TS+

cul-ture at40'Cresultedin a

twofold

increase of 3H

incorporationintoRF DNAoverthatobtained

at350C, whereas incubation of the dnaZts cul-ture at 400C decreased incorporation to less than3% ofthe level in a 350 C control culture (Table 1).

The dnaZ productis, therefore, required for RFreplicationbyboth M13and

OX174.

Requirement for thednaZ function in M13 and gX174 SS DNA synthesis. To investigate the requirement for the dnaZ productin M13 SSsynthesis, F'lac+ derivatives of dnaZts and TS+ cultureswereinfectedat300C andheldat

thistemperaturefor120min, followedby

trans-fer of half of eachcultureto410C. After15min ofadditional incubation, the cultures were la-beled witha 15-min [3Hlthymidinepulse. Incu-bation at 410C enhanced the incorporation of [3H]thymidine into both SS and RF DNAinthe TS+ culture over that obtained at 300C (Fig. 5A), but this treatment dramatically reduced

the amount of incorporation into these DNA

species in the dnaZts culture (Fig. 5B). The small amount of labeled SS and RF in the dnaZts culture thatwasincubated for15min at 410C was completely eliminated by a 30-min incubation period at this temperature. DNA

synthesis in the revertant culture was

un-changed bythis additional incubation at410C

(datanot shown).

Asimilartemperatureeffectwasobserved in

4OX174am3-infected

dnaZts cultures (Table 2). ts and TS+ cultures were infected and incu-bated for45 min at 3000. Half of each culture wasfurtherincubatedateither30 or400Cfor30 min, followed by pulse-labeling with

[3Hlthymidinefor5min. Asaresult of the

30-min incubation at 400C, the number of 3H

countsthatsedimented as SS 4X174 DNA

de-creased, relativetothe300Cincubationdata, by 16%intherevertant culture and by94% inthe dnaZts culture.

It is likely that the terminationofM13 and

OX174

SS synthesis is a direct result ofa

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0-8 16 24 32 8 16 24 32

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FRACTION NUMBER

FIG. 4.Replicationof M13 RF inchloramphenicol-treateddnaZts and TS+ cultures. Theverticalarrows indicatethe position of M13 SS DNA. (A) dnaZts (O) and TS+ (0) cultures wereinfectedas inFig.3 and incubated at35°Cfor 20 minafterthe addition of M13. Thecultureswere then treated withchloramphenicol

(30pg/ml)for 10min,followedbypulse-labelingfor 5 min with[3H]thymidine.(B) dnaZts (@) and TS+ (0)

cultures were transferred to 40°C 10 min after infection at 350C. Ten minutes after the shift to 40°C,

chloramphenicol(30pg/ml)wasadded and the 40°C incubation wascontinuedfor anadditional10min. The

cultures were thenpulse-labeledfor 5 min with[3H]thymidine.

TABLE 1. Replication of Xl74am3 RF indnaZts and TS+ cultures

Total RF I and II synthesis(cpm)

Host

350C

400C

Ratio

40/350C

dnaZts

12,100

300 0.025

TS+ 10,700 22,800 2.1

a C727dnaZts andTS+culturesweregrown to 3

x 108 cells/mlinYET broth (containing 1.5mgof

CaCl2perml)at300C,concentrated10-foldinto 0.01

M MgSO4 and 0.01 M CaCl2, and infected with

OX174am3 (multiplicity ofinfection = 3) at

350C.

After 10min, thecells were diluted 1:10 into YET

broth (plus1.5 mgof CaCl2per ml)containing30

jig

ofchloramphenicolperml andincubatedfor 5 min

at350C.Half of each culturewastransferredto400C,

and theincubationwascontinued for20min. Allof

the cultures were then pulse-labeled with

[3Hlthymidine

for 5 min attheirrespective

tempera-tures,harvested,lysed, andanalyzed.Thedata pre-sented foreach culture are the sum of the

trichloroa-ceticacid-insoluble3Hcountsthatsedimented as RF Iand II DNA.

quirement forthednaZproductinthisreaction rather than asecondary effect ofthe dnaZ re-quirementinRFreplicationwith asubsequent depletion ofanecessaryRFpool. M13 RF repli-cation, butnotSSsynthesis, requiresthednaG product (15);arequirementforthednaC

prod-ucthasbeenobserved in

4X174

RFreplication

but not in SS synthesis (11). In both of these

examples, shifting

phage-infected

temperature-sensitive cultures of these mutantsto nonper-missive temperatures after RFreplication

per-mittedlarge amounts of SSsynthesis thatwere

comparable eitherto thoseobserved in

revert-antculturesatthat temperature(15)or tothose

obtainedinthemutantculturesat apermissive

temperature (11). Shifting M13- or

OX174-in-fected dnaZts cultures to nonpermissive

tem-peratures after RF replication dramatically

curtailedSSsynthesisrelativeeitherto mutant

culturesat a permissive temperatureor to

re-vertantculturesattheelevatedtemperature.

Thus, it appears that the synthesis of M13 and

(PX174

SS DNA requires an active dnaZ product.

DISCUSSION

Basedonthedatainthiscommunication and

those previously reported (10), it appears that

thednaZfunction isrequiredby bothM13and

OX174

in all stages of their DNA replication cycle. Conversion of SSDNA toRF,RF

replica-tion, and the synthesis of progeny single

strands are inhibited in dnaZts mutants at

nonpermissive temperatures. Thefindingthat

anactivednaZproductisdispensableforaloss

in rifampin sensitivity in the M13 SS -- RF

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M13 AND OX174 DNA SYNTHESIS IN dnaZts HOSTS

20

15

10

Ko

04

3

2

24 32 40 48

FRACTION NUMBER

FIG. 5. Synthesis of M13 single-strand DNA in dnaZts and TS+ cultures. F'lac+IH727 dnaZtsand TS+ culturesweregrownto3 x108 cells/ml in YET

broth(containing2pgof thymidineperml)at300C and infected with M13 (multiplicity of infection =

100). Aftera30-min infection period, the cellswere

diluted 1:4 intofresh YET broth (containing 2pgof thymidineperml)andthe incubationwascontinued for 90 min. Theverticalarrowindicates theposition

of M13 SS DNA. (A) TS+ culture. After the 90-min incubationperiod, half oftheculturewastransferred

to410C. Bothsubcultureswereincubatedfor15min

and then pulse-labeled with [3H]thymidine for an

additional15min.Symbols: (0)300C; (a)410C. (B) dnaZts culturetreatedasin(A). Symbols: (0)300C;

(0) 410C.

conversion indicates thatthednaZ function is not neededfor RNA priming andpredicts that it would be required for DNA synthesis on RNA-primed templates. Wickner and Hurwitz have recently demonstrated, by using an in vitro complementation assay, that the dnaZ product is an elongation factor required by

DNApolymeraseIIIforduplexDNAsynthesis

onRNA-primedSSphage templates (21).Inthe

in vitro reaction, using purified proteins, the

dnaZ productwasneeded fornucleotide incor-porationonprimedtemplates byDNA polymer-ase II and III butwasnot absolutely required byDNApolymerase I(21).

Therole of thednaZproductinDNA

polym-TABLE 2. Synthesisof

OX1

74am3

SSDNA in

dnaZts and TS+ cultures

SSDNAsynthesis (cpm)

Host_Host_30°C00 400_40°C Ratio_300C40/

dnaZts 55,600 3,500 0.06

TS+ 66,600 56,200 0.84

a C727dnaZts and TS+cultures were grown and

concentrated as in Table 1 and infected with

4PX174am3(multiplicityof infection = 3) at 300C for

15 min.The cells were diluted1/10 intoYETbroth

(containing 1.5mg ofCaCl2 per ml) and incubated

for 45 min at300C. Half of each culture wasthen

transferred to 40"C, incubation was continued for30

min, andallofthesubcultures werepulse-labeled

with [3H]thymidine for5 min.The3Hincorporation

intoSSDNA wasdeterminedas inTable1.

eraseIII activitydoesnotappear tobeconfined

to parental RF formation. Replication of RF DNA and the asymmetric synthesis of viral single strands, processes requiring an active dnaE product (3, 8, 18), terminate when the dnaZts product is inactivated. As withparental RFformation, however,ourdata donot distin-guish between a requirement for the dnaZ function in ongoing polymerization and in an initiation event in DNA synthesis that may follow RNApriming. 4X174 RF replication and SS synthesis terminate in the absence ofthe E. colidnaG product (12), which has been reported to possess rifampin-insensitive RNA polymer-aseactivity(1). M13 RF replication requires the dnaG product (15) and is also inhibited by ri-fampin (6). M13 SS synthesis does not require the dnaGproduct (15) and is rifampin resistant

for atleasttwosuccessiverounds of SS

synthe-sis (6). It could not be determined, however,

whether a rifampin-sensitive initiation event wasrequired after severalrounds of SS synthe-sis(6).Thus,itispossible that all stages ofM13 and

OX174

DNAreplication require RNA prim-ingevents. Ittherefore follows that the dnaZ product may play a roleinthe initiation of the

DNApolymerizationreaction(e.g., facilitating

the formation of a DNA polymerase/primer complex) rather than being needed for the ac-tualpolymerization reactions involved. The

ob-servation that DNA polymerase III, without

elongation factors, is able to incorporate

nu-cleotides on DNase-treated (21) or gapped

du-plextemplates (22) makes this possibility more attractive.

ACKNOWLEDGMENTS Wethank theindividuals indicated for strains. This work wassupported by American Cancer Society grantNP169and,inpart,by Public Health Service grant 29

VOL. 22, 1977

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A108286 from the National Institute ofAllergyand Infec-tiousDiseases. W.G.H. wassupported by Public Health Service training grants 5T01 GM00337 and 1 T32 GM07126, bothfrom theNational Institute ofGeneral Medical Sci-ences;J.R.W. wassupportedduringaportionof this work by Public Health Service research career development award GM29413 from the sameInstitute.

LITERATURE CITED

1. Boucht,J. P., K.Zechel, and A.Kornberg.1975. dnaG gene product, a rifampicin-resistant RNA polymer-ase,initiates the conversionofasingle-stranded coli-phage DNA to its duplex replicativeform. J. Biol. Chem. 250:5995-6001.

2. Brown, L.R., andC. E.Dowell. 1968. Replicationof coliphageM13. II.Intracellulardeoxyribonucleicacid formsassociated with M13infectionof mitomycin C-treated cells.J. Virol. 2:1296-1307.

3. Dumas, L. B., and C. A. Miller, 1973. Replication of bacteriophage 4X174 DNA in a temperature-sensi-tive dnaE mutant of Escherichia coli C. J. Virol. 11:848-855.

4. Dumas, L. B., C. A. Miller, and M. L. Bayne. 1975. Rifampin inhibitionofbacteriophage 4X174parental replicative-form DNA synthesis in an Escherichia coli dnaC mutant. J. Virol. 16:575-580.

5. Fidanian, H. M., andD. S. Ray. 1972. Replicationof bacteriophage M13. VII. Requirement of the gene 2 proteinforthe accumulationofaspecificRFII Spe-cies.J.Mol.Biol. 72:51-63.

6. Fidanian, H. M., and D.S. Ray. 1974. Replicationof bacteriophage M13. VIII. Differential effectsof rif-ampicinand nalidixic acidonthesynthesisofthetwo strandsof M13duplexDNA. J. Mol. Biol. 83:63-82. 7. Geider, K. 1976. Molecular aspects of DNAreplication

inEscherichia coli systems. Curr.Top. Micro. Immu-nol. 74:56-112.

8. Greenlee, L. L. 1973.Replicationofbacteriophage OX-174 ina mutant ofEscherichia coli defective in the dnaE gene. Proc. Natl. Acad. Sci. U.S.A. 70:1757-1760.

9. Guthrie, G. D., and R. L. Sinsheimer. 1963. Observa-tions on the infection ofbacterial protoplasts with deoxyribonucleic acid ofbacteriophage OX174. Bio-chem.Biophys. Acta72:290-297.

10. Haldenwang,W.G.,andJ. R.Walker. 1976. Parental RFformation ofphageqbX174 and M13requiresthe dnaZ geneproductofEscherichia coli. Biochem.

Bio-phys.Res. Commun. 70:932-938.

11. Kranias,E.G.,and L. B.Dumas.1974.Replicationof

bacteriophage 4X174 DNA in a temperature-sensi-tivemutantofEscherichia coli C. J. Virol. 13:146-154.

12. McFadden, G.,and D. T.Denhardt.1974.Mechanism ofreplicationof4X174 single-strandedDNA. IX. Re-quirementfor theEscherichia coli dnaGprotein. J. Virol.14:1070-1075.

13. Marvin,D.A.,and B.Hahn. 1969. Filamentous bacte-rial viruses. Bacteriol. Rev. 33:172-209.

14. Ray,D.S. 1970.Replicationofbacteriophage M13. IV. Synthesis of M13-specific DNA in the presence of chloramphenicol.J. Mol. Biol.53:239-250.

15. Ray,D.S.,J.Dueber,andS.Suggs. 1975.Replication

ofbacteriophageM13. IX.Requirementof the Esche-richia colidnaG function of M13 duplex DNA replica-tion. J.Virol.16:348-355.

16. Schekman, R.,A.Weiner,andA.Kornberg. 1974. Mul-tienzyme systems of DNA replication. Science 186:987-993.

17. Sinsheimer,R. L. 1968. Bacteriophage qX174 and re-lated viruses. Prog. Nucleic Acid Res. Mol. Biol. 8:115-169.

18. Staudenbauer,W. L. 1974.Involvement of DNA polym-erasesI and III in the replication ofbacteriophage M13. Eur. J.Biochem. 49:249-256.

19. Taketo,A. N975.Replication ofOAandOX174in Esche-richia coli mutantsthermosensitive in DNA synthe-sis. Mol. Gen. Genet. 139:285-291.

20. Truitt, C. T. and J. R. Walker. 1974. Growth ofphages X,4X174and M13requiresthe dnaZ geneproduct of Escherichia coli. Biochem. Biophys. Res. Commun. 61:1036-1042.

21. Wickner, S., and J. Hurwitz. 1976. Involvement of Escherichia coli dnaZ gene product in DNA elonga-tion in vitro. Proc. Natl.Acad. Sci. U.S.A. 73:1053-1057.

22. Wickner, W., R. Schekman, K. Geider, and A. Korn-berg. 1973. A new form of DNA polymerase III anda copolymerase replicate a long single-stranded primer-template. Proc. Natl. Acad. Sci. U.S.A. 70:1764-1767.

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