Replication of bacteriophage M13 IX. Requirement of the Escherichia coli dnaG function for M13 duplex DNA replication.

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Replication

of

Bacteriophage M13

IX. Requirement

of the Escherichia coli dnaG Function for M13

Duplex

DNA

Replication

DAN S. RAY,* JEANENE DUEBER, AND SIDNEY SUGGS

MolecularBiology Institute and Department ofBiology,* University of California, LosAngeles, California

90024

Received for publication 3 March 1975

Temperature-shift experimentswithanEscherichia coli dnaG strain indicatea

requirement for the dnaG function for M13 phage production only at an early

stageofinfection. Mutant cells infected atnonpermissive temperature formthe parental RF (SS -- RF) but do not replicate further. A shift to nonpermissive temperature after infection inhibits RF RF replication but not RF SS

synthesis. Thesynthesis ofboth strands of the duplexRF wasinhibited equally

after a temperature shift during RF -- RF replication. We infer that thednaG protein is required for M13 production only during RF replication and that it is required for thesynthesis ofboth strands of the RF.

Bacteriophage M13 contains fewer than 10 genes and consequently utilizes many of its host's functions initsreplication(10). With the

availability of many bacterial mutants defec-tive in their own DNA replication, it is now

possibletodetermine which of the known Esch-erichia coli genes involved in bacterial DNA replication also function in M13 DNA

replication. E. coli polA mutants defective for dnaA, dnaB, dnaC(D), dnaE, or dnaG are all blocked in M13 production at nonpermissive temperature (11). Arecentlydescribed mutant,

dnaZ, also fails to support M13 replication at high temperature (18).

Studieswith dnaB and dnaEmutants(12, 17) suggest that neither of these genes are

abso-lutely essential for theinvivo conversion ofthe

M13 viral DNA to its replicative form

(SS

RF). However, both ofthese gene products are

required for later stages of replication-the dnaBproduct

being

required forRF replication (RF RF) but not for single-strand synthesis (RF SS) and the dnaE product required for RF replication and possible for single-strand synthesis aswell. The dnaA gene appears to be required for oneofthe final steps of replication, the closure of linear viral strands (4). At nonper-missive temperature this mutant accumulates linear rather than circular viral strands.

The dnaG gene has been implicated recently inthe initiation of Okazaki fragments (8) and in the formation ofanRNA primer forthe comple-mentary strand during parental RF formation invitroby G4 (J. P. Bouche, K. Zechel, and A.

Kornberg, J. Biol. Chem., in press; K. Zechel, J. P.

Bouche,

and A.Kornberg, J. Biol. Chem., in press), a 4X-related bacteriophage. This stageof 4Xreplicationas well as RFreplication andsingle-strand synthesis are all inhibited in vivo in a dnaG mutant at nonpermissive tem-perature (9).

Our studies indicate a requirement for the dnaG geneproductinonlyasingle stage ofM13 replication-the replication ofRF. Neither pa-rental RF formation (SS - RF) nor

single-strand

synthesis

(RF - SS)

requires

this host function.

MATERIALS AND METHODS Phage and bacterial strains. M13wild-type and M13 5amH3 are from the collection ofDavid Pratt and have been described previously (13, 14). E. coli PC3(F-, leu-, thy-,strR,dnaGt)waskindly provided by Randy Schekman. (We have confirmed the

loca-tion of the temperature-sensitive locus by genetic

mapping ofthe mutation.) E. coli PC3X8 is an F+

derivative of PC3. PC3X8 rev. is a

temperature-resistant revertant ofPC3X8which forms colonies at

41 C; however, the reversion of the dnaGt' locus is

only partial sincePC3X8 rev. neither formscolonies

norsynthesizesDNA at 42C.E.coliK37 (supD) was used as indicator for M13 in plating experiments.

Phage were titered by the agar overlay method

described byAdams (1).

Media. Bacteria were grown in glucose-Casamino

Acids medium (16)containingthymidineat 2

,g/ml.

For radioactive labeling of DNA the medium was

supplemented with [3H]thymidineat 10MCi/ml.

Preparationof39P-labeledphage. Forthe

prepa-rationof32P-labeledM13,E. coli K37 wasgrown in 50

348

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resuspended in 0.01 M Tris (pH 8.0) containing 1 mM EDTA and0.5%Sarkosyl and precipitated a second timewithpolyethylene glycol and NaCl. This

precipi-tate wasfinally resuspended in 1 ml of 0.01MTris (pH

8) containing 1 mM EDTA andsedimented for 16 h througha 5 to20%sucrosegradientat24,000 rpmand

5Cin aBeckmanSpinco SW27rotor.Fractions were collected from the centrifuge tube at the end of the

runandassayed for both 32P and PFU.

Harvest and lysis technique. Isotope incorpora-tionwasterminatedbypouring infected cultures into

anequal volume of ice coldTEN-CN buffer (0.01 M Tris, pH 8.0, 1mMEDTA, 50 mM NaCl, and 10 mM NaCN). The cells were harvested and washed by centrifugation for 1 min at 15,000 rpm in aSorvall SS-34rotor at 4C and resuspension in 1 ml ofcold TEN-CN buffer. After washing and resuspending the cellsasecondtime, the cellswerelysedby adding100 Mloflysozyme (4 mg/ml inwater), incubatingfor10

min at 37C, adding 100

Al

of 10% Sarkosyl, and incubatingforanadditional 10min at 37C. Carewas

taken not to shear the viscous lysate during the addition andmixingoftheSarkosyl.Thiswas

accom-plished by gently pipetting the detergent into the lysateand slowly rollingthe tubebyhand until well mixed.

Neutral velocity sedimentation oflysates. The viscouslysatecanbegently pouredontothe top ofa5

to 20% sucrose gradient for sedimentation analysis.

Pipetting such lysates should by avoided since this leads to breakage ofthe bacterial DNA and

conse-quently an increased background of bacterial DNA sedimenting in the region of the small phage DNA species. Neutralgradientscontained 0.01 MTris(pH 8), 1mMEDTA, and 1.0 MNaCl. Sedimentationin

an SW27ultracentrifuge rotor wasfor 17hat23,000 rpmand 5C. Fractionswerecollected from the topby pumping 50%sucroseintothe bottom of thegradient and collecting the fractions through a specially de-visedadaptorplacedon topof thecentrifugetube.

Alakline centrifugation techniques. Alkaline equilibrium centrifugation was performed as previ-ouslydescribed (5, 15).

Radioactive counting. Aliquots from neutral

su-crose gradient fractions were spotted directly onto

Whatmanno.3filter papercircles,dried,andcounted by liquid scintillation countinginaBeckman LS230

two-channel counter. Fractions from alkaline CsCl gradientswerecollecteddirectlyontoWhatmanno.3

filter papercircles, dried,andcountedasabove.

proachesthatof theculture heldat 32 Cforthe

entire time (Fig. lb). We infer from this

obser-vationthatthe dnaGfunction isrequiredforSS

-RF

and/or

RF - RF

replication

but not for

RF- SSsynthesis, the finalstageofviral DNA

synthesis.

Parental RF formation (SS - RF) in

PC3X8 dnaG cells. The first stage of replica-tion of M13

DNA,

the conversion of the

in-fecting viral strandtothe parental RF, doesnot

require the synthesis ofviral proteins. Thisset

of reactions is readily carried out in crude extractsofuninfectedcells andcanbe

reconsti-tuted in vitrousing

purified

bacterial proteins (6). To investigate thepossible role of the dnaG protein in this stage of replication in vivo we

have analyzed RF molecules formed in E. coli PC3X8

dnaG

atdifferenttemperatures. Figure

2 shows the neutral velocity sedimentation profiles Jf DNA from lysates of cells infected

at32, 39,and41C. The cellswereinfectedwith 32P-labeled M13 in growth medium containing

[8H ]thymidine,

harvested at5 min after infec-tion,

lysed,

and sedimented

through

neutral

sucrose

gradients.

Since the

parental

RF

con-tains 32p label from the infecting viral

strand,

theamountof3 pfoundin

replicative

forms isa

direct measure of the amount of

parental

RF formed. More

parental

RF is formedatboth39

and41

C

thanat32C

(Fig. 2). Similar

amounts

of

parental

RFwereformed

(data

not

shown)

in

a revertant of the dnaG mutant strain and in twodifferent

wild-type

strains,

indicating

alack of a requirement for the

dnaG

function in

parental

RFformation.

Replication

beyond

this initialstage of

repli-cationwasinhibitedat39and41

C

asshown

by

the incorporation of 3H label into

only

the

complementary strand of RF II molecules

(Fig.

3). Extensive RF

replication

would have

re-sultedinthe

labeling

ofboth strandsoftheRF.

Even asingle roundof

duplex

DNA

replication

would have led to an

easily

detectable amount of 3H in the viral strand ofprogeny RF

mole-cules, unless the progeny RF molecule

having

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350 DUEBER, ANDSUGGS

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dnoG40'

KC8

30 60 90 120 0 30 60 90 120 150

time(min)

FIG. 1. Requirement for the dnaGfunction early in M13replication.Culturesof E. coli PC3X8 dnaG and E. coliPC3X8 rev. were each grown in glucose-Casamino Acidsmediumat 32Cto acell density of2x 108/mland infected with M13 atamultiplicity of infection of I to 2. (a) Phage productionincultures either held at 32 Cor

shifted to 40 C at 10 minprior to infection; (b) phage production in cultures either held at 32 C or shifted to

40 C at 40 min after infection. Symbols: circles, PC3X8 dnaG; triangles, PC3X8 rev.; open symbols, 32 C; closed symbols, shiftedto 40C.

theparental viral strand isselectively retained

inthe RF II form while the otherdaughter RF is closedtoform anRFI. Should such a

preferen-ial closure occur, it would be possible for a

single

round of RF - RF

synthesis,

butnomore, to go undetected by this method of analysis. Whether or not preferential closure of RF II molecules might occur, extensive RF replication does not take place in the dnaG mutant at nonpermissive temperature.Theobserved block

to RF - RF

synthesis

but not to SS - RF

synthesis indicated that the dnaG function is required only after the initial formation of the

parental RF.

Since parentalRF formationmight

conceiva-blyinvolveresidual activity ofthe dnaG protein it was ofinterest to investigate this reaction in cells preincubated fora longertime at nonper-missive temperature. Figure 4 shows the sedi-mentation profile of DNA from a lysate of PC3X8 cells infected with unlabeled M13 at 40 minafter transfer from 32 to41 C. The amount of3H-labeledRFobserved here is similar to that found in cells infected at only 8 min after transfer(Fig. 2c). These results strongly support the lack ofa requirement forthe dnaG protein in the SS - RF conversion in vivo.

Requirementof the dnaGproteinfor RF RF replication.Toexplorefurther the

involve-ment ofthe dnaG

protein

in RF - RF

replica-tionwe have investigated the temperature

sen-sitivity

of RF - RF

synthesis

in mutant and revertant cells infected with M13 mutants de-fective in gene5.RF RFsynthesis takes place

in the absence of the gene 5 DNA-binding

protein

but not that of RF - SS (13).

Figure

5 shows the neutral velocity sedimentation

pro-files oflysates of PC3X8 and a

partially

tem-perature-resistant revertant ofthe same strain. Both strains were infected with M13

5amH3,

and portions were transferred to separate

cul-ture tubes at 32, 39, and 41 C at 15 min after infection. Each culture was pulse labeled with [3H]thymidinefrom 25to 30minafterinfection, harvested, lysed, and sedimented through neu-tral sucrose gradients. Labeling of replicative forms wasinhibitedat39and41 Cin thednaG mutantstrainbut less sointhe revertant strain. These results directly implicate the dnaG pro-tein in M13 RF , RF replication.

To investigate the possible selective inhibi-tion ofthesynthesisofthe M13 complementary strand we have analyzed the distribution of label between the two strands of RF II mole-cules formed in the dnaG mutant at 32 and 39 C. Separationofthe twostrands is achieved

by equilibrium centrifugation in alkaline CsCl. Figure 6 shows the 3H label contained in the

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0

E

-r)

0 20 30

Fraction No.

0

c

0

a-rn~ E

C-)

0

10

20

30

40

50

[image:4.503.261.445.53.313.2]

3 Fraction No

FIG. 3. Distribution of 3H label between the viral

~

2 andcomplementarystrandsofRFlIIformedinPC3X8

dnaGat39and41 C. Pooled fractionsfrom theRFII

regions of Fig. 2b andc weredialyzed against0.01M t4

~

Tris(pH8) containing 1 mMEDTA and then

sedi-mented to equilibrium in alkaline CsCI gradients.

Densityincreases from righttoleft in these gradients.

0 Thepositions in thedensitygradientof viral strands

40 50 (VS) and complementary strands (CS)areindicated

by verticalarrows.Symbols: 32pJ;0,3H.

FIG. 2. Parental RFformation(SS RF)inPC3X8

dnaG. Three 20-ml cultures of PC3X8 dnaG were

growninglucose-CasaminoAcidsmedium at32Cto

a celldensity of approximately2x 108cells/ml. Two

of the cultures were transferred to39and41 C, and after 7 min [3H]thymidine was added to all three

cultures. At 8 min after the temperature shift each

culturewasinfected with 32P-labeledM13at1010/ml,

and after an additional 5 min all cultures were harvested, lysed, and sedimented through neutral

sucrosegradientsasdescribed. The direction of

sedi-mentation is indicated by the horizontalarrow. The

sedimentationpositions of RF I,RFII,andphageare

indicatedbyverticalarrows.Symbols: ,32p;0,3H.

separated strands of the RF II's. The extentof labeling of the complementary strand relative

tothatoftheviralstrand is thesameat39 Cas

at 32 C. Adifference in the relative labelingof thetwostrandswould have beenexpectedifthe viralstrand hadbeen synthesizedpreferentially

at the nonpermissive temperature. This evi-dence suggests that the dnaG protein is not

required exclusively for the synthesis of the complementary strand of the RF duringRF ,

0

LO

10-

5-

0-10 20 30 40 50

Fraction No.

FIG. 4. Parental RF formation in PC3X8 dnaG

after extendedincubationat41 C.A40-ml culture of

PC3X8 dnaGwasgrownat2 x 108cells/mlasinthe

legendtoFig.2.The culturewasshiftedto41 Cfor40

min before infection byunlabeledwild-typeM13. One

minutepriortoinfection [3H]thymidinewasaddedto

the cultureto 10 MCi/ml. At 5minafter infection the

culturewasharvested, lysed,and sedimentedthrough

aneutralsucrosegradientasdescribed.The direction

ofsedimentation isfrom lefttoright.

RF replication.

Single-strand synthesis

(RF

SS) in

PC3X8 dnaG. The final stage of M13 DNA

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352 RAY, DUEBER, ANDSUGGS

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F

>(b)39

--b

(e)

390

E

'N'

(c)410

(f)410

5

l0 20 30 40

l0

20 30 40

[image:5.503.108.410.70.439.2]

Fraction

No.

FIG. 5. RFreplication(RF , RF) inPC3X8 dnaG andPC3X8rev. Three 30-mlculturesof PC3X8dnaG

andthree 30-ml cultures ofPC3X8rev. wereeach grownat 32 C ingiucose-CasaminoAcids mediumtocell densitiesof approximately2 x 108/mland theninfectedwith M13 5amH3 at1010/ml.At 15min after infection

twoculturesofeachsetweretransferredto39and41 C,andafter10min each culturewaspulselabeled with [8H]thymidinefor5min. All cultures wereharvested, lysed,and sedimented through neutralsucrose gradients

asdescribed. The direction of sedimentation is indicated by the horizontal arrows.

replication, the asymmetric

synthesis

of viral

strands, requires two viral gene functions in addition to host functions

(13).

To

investigate

the potential role of dnaG in this stage of

replicationwehavepulselabeled both thednaG

mutantand itsrevertantaftershiftingto39and 41 C at 45 min after infection. The cells were

pulse labeled with [3H]thymidine for 5 min at

10 min after the temperature shift. Figure 7 shows the velocity sedimentation pattern of

lysates fromeach of the infected cultures.

Sub-stantial amounts of viral single strands were

synthesized even at 41 C in the dnaG mutant (Fig. 7c). Comparison with the amount of

syn-thesisobservedintherevertantstrainshows no significant differenceinsingle-strand synthesis. We infer from thisthatasymmetricviralstrand

synthesis does not require a functional dnaG

protein for continued synthesis.

DISCUSSION

The observations presented here indicate that the E. colidnaGfunction is required for M13 RF replication but not for parental RF formation

(SS , RF) or

single-strand synthesis

(RF

SS). A point of concern with regard to the significance of these results for the SS - RF reaction is the need for only a single round of

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20

3

0

E",

N~~~~~~~~

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10 20 30 40

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No.

FIG. 6. Distribution of 3H label between the viral and complementary strands ofM13 5amH3 RF II formed at 32 and 39 C in PC3X8 dnaG. Pooled fractions from the RF II regions of Fig.5aand bwere dialyzed and sedimented to equilibrium in alkaline CsCI as in the legend to Fig. 3. 32P-labeled viral DNA was added as a density marker. Density

in-creases from right to left. The positions of viral

strands (VS) and complementary strands (CS) are

indicatedby verticalarrows.Symbols: 0,3p;0,3H.

replication for parental RF formation tooccur.

This small amount of synthesis might readily takeplace duetoeitherresidual enzyme

activ-ityatthenonpermissivetemperatureor reratu-ration of the enzyme during some stage of processing of the infected cells prior to lysis. Thestrongest argumentagainst suchanartifact

isthe clean inhibition ofRF RFreplication in cells infected at nonpermissive temperature. Had there been a similar requirement for the dnaG function for the SS RF reaction then thereshould have beenasignificant decreasein

the amount of parental RF formed at high temperature. Yet, just the opposite was

ob-served. There was an actual stimulation of

parental RF formation at temperatures of 39 and 41 C. This lack of a requirement for the

dnaG protein for the conversion ofM13 SS to

RF agrees with the observations made on in

vitro systems (6, 21). This reaction has now

been reconstituted from purified proteins and requires only RNA polymerase, aDNA

polym-erase III holoenzyme, and DNA unwinding

protein (7).

strandsofthe RFindicatedarequirement of the

dnaG function for thesynthesisofboth strands. Residual labeling ofRF molecules in the dnaG

mutant at high temperature labeled both strands equally. In parallel experiments with a

dnaB mutant (unpublisheddata) we alsofound equal inhibition ofsynthesis of bothstrands of

M13 RF at nonpermissive temperature. We therefore infer that neither the dnaB nor the dnaG proteins are required solely for

comple-mentarystrandsynthesis during RF replication.

ThednaGproteinhasbeen shownrecently to

be a rifampicin-resistant RNA polymerase

ca-pable offorming anoligoribonucleotide primer on single-stranded G4 DNAcoated withthe E.

coli unwinding protein (Bouche et al., J. Biol.

Chem., inpress).Atranscriptof aunique region

of the G4 DNA can serve as a primer for the

synthesis

ofthe G4 complementary strand dur-ing

SS

_RFsynthesis. This function is similar

tothatofRNApolymeraseininitiatingthe M13 complementary strand (4, 6, 20). Ittherefore is

likely that the dnaG protein might prime the synthesis of oneof the strands of the M13 RF. However, should the

dnaG,

protein be involved in the initiation of either strand of the RF

during

RF - RF

replication,

the mechanism of initiation would needtobeunique tothatstage of replication since neither complementary strand initiationduring SS - RF

synthesis

nor

viralstrandinitiationduring RF - SS synthesis require the dnaG function. It is unlikely that thednaGproteinisdirectly involvedin

comple-mentary strand initiation since rifampin

selec-tively

inhibitsM13

complementary

strand

syn-thesis during RF - RF

synthesis

(4). This

observation suggests

that,

asfor M13 SS- RF

synthesis, the

complementary

strand

primer

is formed by RNA polymerase.

We are thus left with the

possibility

thatthe

dnaG protein initiates viral strand

synthesis.

Inhibition ofviral strand synthesis

might

also prevent complementary strand initiation since RF - RF replication appears to occur

by

a

rolling-circle mechanism (7) inwhich the viral

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354 RAY, DUEBER, AND SUGGS

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lO0(b)

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20

30

40 l0

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FIG. 7. Single-strandsynthesis (RF SS)in PC3X8 dnaG and PC3X8rev. Three30-ml culturesof PC3X8 dnaGandthree 30-ml culturesofPC3X8rev.wereeach grownat32C inglucose-CasaminoAcids mediumto a

celldensity ofapproximately2x 108/ml.At45minafter infectiontwoculturesofeachset weretransferredto39

and41 C, andafter 10mineach culturewaspulse labeled with [3H]thymidinefor5min. All cultures were

harvested, lysed, andsedimentedthroughneutralsucrosegradientsasdescribed. The directionof sedimenta-tion is indicatedbythehorizontalarrows. Thesedimentationpositions ofRFI,RFII, and SSareindicatedby

verticalarrows.

strandis theelongatedstrand(19).Inthiscase,

the templateforcomplementarystrand synthe-sis, the elongatedtail of the viralstrand, would

notbeformed and thesynthesisofboth strands would be inhibited equally. The experiments presented herewouldthereforenotdiscriminate between a direct requirement of the dnaG

protein for the synthesis of both strands of the RForofonlythe viral strand. Should the dnaG

protein prove to be directly involved only in

viral strand synthesis during RF RF

synthe-sis, it will be of interestto comparethe

mecha-nism ofinitiation of viral strands during both

RF RF and RF SSsynthesis. From results

presented here, we infer that the dnaG protein

is notrequired forviralstrandsynthesis during

RF SS synthesis.

ACKNOWLEDGMENTS

This research was supported by Public Health Service

grant AI-10752 from the National Institute ofAllergy and

InfectiousDiseases.

LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages. Interscience Pub-lishers, NewYork.

2. Bouvier,F., andN.D. Zinder. 1974.Effects ofthe dnaA thermosensitive mutation ofEscherichia coli on bac-teriophage fl growth and DNA synthesis. Virology 60:139-150.

3. Brutlag, D., R. Schekman, and A. Kornberg. 1971. A possible roleforRNA polymerase inthe initiation of M13 DNAsynthesis. Proc. Natl. Acad. Sci. U. S.A. 68:2826-2829.

4. Fidaniain, H. M., and D. S. Ray. 1974. Replication of bacteriophage M13. VIII. Differential effects of rif-ampicinand nalidixic acid onthesynthesisofthetwo strands of M13duplexDNA. J. Mol.Biol.83:63-82. 5. Francke, B. F., and D. S. Ray. 1971. Formation of the

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OX-174and initial events in itsreplication.J Mol. Biol. 61:565-586.

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rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 33:824-835.

8. Lark,K.G.1972.Geneticcontrol over the initiation ofthe

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13. Pratt, D., and W. S. Erdahl. 1968. Genetic control of bacteriophage M13 DNA synthesis. J. Mol. Biol. 37:181-200.

14. Pratt, D., H. Tzagoloff, and W. S. Erdahl. 1966. Condi-tional lethal mutants of the small filamentous- coli-phage M13.I.Isolation, complementation, cell killing,

time of cistron action. Virology 30:397-410.

15. Ray, D. S. 1969. Replication ofbacteriophage M13. II.

The roleofreplicative formsinsingle-strand synthe-sis. J. Mol.Biol. 43:631-643.

16. Ray, D. S., and R. W. Schekman. 1969. Replicationof

RNA polymerase from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:4425-4428.

21. Wickner, R.B.,M. Wright, S. Wickner, and J. Hurwitz.

1972. Conversion of 6X174 and fd single-stranded DNAtoreplicativeforms inextractsofEscherichia coli. Proc. Natl.Acad. Sci. U.S.A. 69:3233-3237.

22. Yamamoto, K. R., B. M. Alberts, R. Benzinger, L. Lawhorne, and G. Treiber. 1970.Rapidbacteriophage sedimentation in thepresence ofpolyethylene glycol and its application to large-scale virus purification. Virology 40:734-744.