Synthesis of complex forms of bacteriophage phiX174 double-stranded DNA in a temperature-sensitive dnaC mutant of Escherichia coli C.

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Copyright ( 1975 AmericanSocietyforMicrobiology Printed inU.S.A.

Synthesis

of

Complex Forms of Bacteriophage iX174

Double-Stranded

DNA in a

Temperature-Sensitive

dnaC

Mutant of

Escherichia coli C

EVANGELIA G. KRANIAS1 AND LAWRENCE B. DUMAS*

DepartmentofBiochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201 Received forpublication3March 1975

Fast-sedimenting forms of bacteriophage kX174 double-stranded replicative-form DNA observed in normal infections continued to accumulate at the nonpermissive temperature in a temperature-sensitive dnaC mutant of Esche-richia coli. These complex molecules accounted for up to half of the DNA

synthesized duringshortpulses at the nonpermissive temperature. Theywerethe

dead-end products ofDNA synthesis, not intermediates in normal

replicative-form replication. The data suggest that these

higher-than-normal-molecular-weight DNA moleculesresult from abnormal initiationof kX174replicative-form DNAreplication.

Two to four percent of the intracellular dou-ble-stranded replicative-form (RF) of bacterio-phage

OX174

DNA are multiple-length mole-cules (1, 11, 15, 16). Mostofthese are multiple-length continuous circles, although interlocked monomer circles are also observed. Both kinds ofstructures arefound to be theproduct of both DNA replication and DNA recombination. However, interlocked monomer circles are the predominant product of the DNA

recombina-tion pathway, and multiple-length continuous

circles are thepredominantproductofthe DNA replication pathway (1).

Suchcomplex forms ofcircular DNAare not

unique to viruses. Multiple length forms of

bacterial plasmidDNAhave also been observed

(7, 8, 9, 10).

Wehave observed the accumulation of higher-than-normal-molecular-weight forms of

OX174

double-stranded DNA in a temperature-sensi-tive dnaC mutant of Escherichia coli at both the permissive and nonpermissive temperature forthe dnaCprotein activity. The dnaC protein isspecifically -equired for the initiation of DNA synthesis (2, 17). These complex forms of

OX174

RF DNA were also synthesized in the

parental host strain, but not at the

nonpermis-sive temperature in a dnaEmutant of the same parental host. The dnaE gene product, DNA polymerase III, is essential for DNA chain elongation (5, 14). We conclude that complex

DNA molecules result fromabnormal initiation

of DNAsynthesis. The conditions under which these molecules accumulate are reported, and Present address: Department of Biochemistry. North-westernUniversity Medical School, Chicago,Ill.60611.

their relationship to the complex

OX174

RF DNA molecules observed by others in normal infections is discussed.

MATERIALS AND METHODS

Bacteria and phage strains. LD301 and LD331 are,respectively,dnaEts,anddnaCtsmutants of H502 (uvrA-, thyA-, endI-) previously described (4, 12). kX174am3(gene E)is alysis-defective mutant.

Infection and preparationof cell lysates. These procedures have been described (4). In these

experi-mentsphage infection wascarriedoutinmedia

sup-plemented with 0.01

lsg

ofthymineperml. This low

concentration of thymine was used to increase the

specific activity of [3H]thymidine-labeled

intracel-lular OX174DNA. In0.01jig ofthymineper ml

un-infected host cellsdouble in number before

multipli-cation ceases. The yield of kX174 am3 in this

concentration of thymine is 15% of that in 2 ug of

thymine per ml. The replication of OX174 am3 RF

DNA in the dnaC mutant host is as

temperature-sensitive in 0.01 ug ofthymineperml as in 2 ggper

ml.

Centrifugation analyses. The conditions of the

zone sedimentation analyses in neutral pH sucrose

have been described (12). Alkaline pH sucrose

gradi-entsconsisted of 5 to20%sucrose in 0.2MNaOH,0.8

MNaCl, 2mM EDTA, and0.1% Sarkosyl, pH 12.6.

Cesium chloride density gradient analysis was

carried out as describedbyMuller-Wecker et al. (13).

Thesampleswerespun in a Beckmantype 65 rotor at 45,000 rpm for 48 h at 20 C.

Electron microscopy. The intracellular phage

DNA wasextractedbyusing theprocedureofGodson

and Vapnek (6). The DNA was fractionated on

neutral sucrose gradients, dialyzed against 50 mM

Tris-hydrochloride, 1 mM EDTA, pH 8.1, at 4C,

and precipitated at -20C by addition of 2 to 3 volumes of ethanol.

412

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SYNTHESISOF COMPLEX

OX174

DNA 413

The DNA sampleswere spread and shadowed by using thetechniquesdescribed by Davis et al. (3).

Chemicals. [Methyl-H

lthymidine,

56 Ci/mmol,

was purchased fromAmersham/Searle. Mitomycin C,

chloramphenicol, egg white lysozyme, and protease (type VI) werepurchased from Sigma Chemical Co.

RESULTS

Synthesis of abnormally

high-molecular-weight

qX174

RFDNA. Previous experiments

(12) showed that one of the major products of

DNA synthesis in a

4X174-infected

dnaCt8

mutant host at 41 C, during the double-stranded RF DNA replication stage, was a

species that sedimented in a broad band at

about the same rate as

kX174

single-stranded

DNAin high ionic strength medium (27S). We

monitored the synthesis of this DNA under

conditions where the specific activity of the

radioactive label was increased, and examined

its structure.

To determine whether this DNA was

single-stranded

phage

DNAora

high-molecular-weight

form of

4X174

double-stranded DNA, liquid

cultures of the

dnaCts

mutant LD331 were

infected with

OX174

am3 inthe presence of 30

,ug of

chlorampenicol

perml. Thisconcentration

of chloramphenicol inhibits single-stranded

synthesis,

allowing

prolonged RF

replication

(18).The

phage

DNAinthese cellswaslabeled

with

[3H]thymidine

at30and41 C. 3H-labeled

DNA that sedimented faster than the

single-stranded

phage

DNAmarkerwas seenin

lysates

ofthe infected cells

pulse-labeled

at30 C

(Fig.

1A), and pulse-labeled after shifting to 41C

(Fig. 1B). The normal products of

OX174

RF

DNA replication, RFI (21S) and RFII (16S),

were also observed. The 3H-labeled

fast-sedi-menting DNA was also observed after

longer

pulses, and after chases at 30 and 41 C (see

below).

The DNAinthe

fast-sedimenting

bandsfrom

sucrose

gradients

was

subjected

toequilibrium

buoyant density analysis (Fig. 2).

Almost all of

the DNA in this band from

lysates

of cells

labeledateithertemperaturehad the

density

of

double-stranded DNA. The same was true of

the

fast-sedimenting

DNA from

lysates

ofcells

that had been chased at both temperatures

(data not shown). It was not an aggregate of

normal

OX174

RF DNA held together by pro-teins since it was stable to

degradation

by

nonspecific proteases followed

by

heating 20

min at 56C (Fig. 1).Itwas nothost DNA since

no radioactively labeled DNA

sedimenting

at

the same rate in neutral

pH

sucrose

gradients

wasfoundin extractsofuninfected host cellsat

30 or 41 C in identical experiments

(data

not

shown).

We conclude

therefore,

that a

higher-6

4

60-to I

a-

0

z

%WOO

2

0

6

4

2 o)

-%f

10

20

30 FRACTION NUMBER

FIG. 1. Zone sedimentation of intracellular phage DNA from 4X174 am3-infected LD331. A liquid

culture of bacteria was grown at 30 C to a cell density of3 x 108 cells/ml on TPGA medium sup-plemented with 2

jug

of thymine per ml. The cells were collected by centrifugation and suspended in

0.1volume of TPG medium. Mitomycin C was added

to0.1 mg/ml.After 20min in the dark the cells were collected and resuspended in 1 volume of TPGA medium supplemented with 0.01

Ag

of thymine per ml. Phage (10 per cell) and chloramphenicol (30

,g

per ml) were added at zero time. After 55 min at

30 C 200MCiof [3H]thymidine were added to 20 ml of the infected culture. Two minutes later this portion of the culture (A) was rapidly chilled. At 55 min after

infection another 20-ml portion of the culture was

shifted to 41 C. Fifteen minutes later 200 ,uCi of

['H]thymidinewere addedfor 2 min, and the culture

was rapidly chilled (B). The chilled cells were col-lected, washed, and lysed. Thelysates were digested with protease, heated 20 min at 56 C, and sedi-mentedthrough neutral pH sucrose gradients (16 h at

4 C at 25,000 rpm in a Beckman SW27 rotor). Thirty fractions were collected from the bottom of each gradient. The amount of radioactivity in 0.2

ml of each fraction was measured. Arrows indicate the positions of added marker "2P-labeled virus

DNA.

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6 B

06

02

10

40 111,

60 -C

40

20

40

60 FRACTION

NUMBER

FIG. 2. Cesium chloride equilibrium buoyant den-sity analysis of the fast-sedimenting OX174 DNA.

TheDNA inthefast-sedimenting bands from neutral

pHsucrose gradients was dialyzed, concentrated by

alcohol precipitation, and spun in CsCI gradients. 32P-labeled

kX174

single-strandedDNA markerwas

added to each sample. Frame A represents the banding profile of the DNA from a lysate of kX174

am3-infected LD331 treated exactly asdescribedfor sampleA inFig. 1. FrameB represents the banding profile of the DNA from a lysate of infected cells

labeled for 30min at 41 Cbeginning at 5 minafter

the temperature shift-up. Frame C represents a

than-normal-molecular-weight form of

OX174

double-stranded DNA was synthesized in this

mutant hostboth at 30 and 41 C.

Theamount ofhigh-molecular-weight kX174

DNA synthesized in LD331duringshortpulses

at 30 Cwasaboutthe same in mediacontaining 1 and 0.01

Ag

of thymine per ml. This DNA represented 11 +3and16 ±3%, respectively,of the iX174 double-stranded DNAsynthesizedat these thymine concentrations. Similarly, this

complex DNA was observed in

OX174-infected

LD331 thathad notbeen treatedwith mitomy-cin C. It is not therefore an artifact of these experimental conditions.

In experiments similar to those described in Fig. 1, we measured the amounts ofthe

abnor-mally high-molecular-weight,

OX174

double-stranded DNAsynthesized at30and41 C, and the rates of synthesis of the

high-molecular-weightand normal RF DNAat 41 C relativeto

those at 30C. The data from threeexperiments

issummarizedinTable1. These data show that

the percentage of high-molecular-weight DNA

synthesized during short pulses at 30C was

about the same in LD331 and the parent host

strain H502. The rate of normal RFreplication at 41 C in LD331 was about 70% less than at 30 C. The rate ofsynthesisofthe

high-molecu-lar-weight

OX174

DNA in both hosts at 41 C

was at least ashighasthatat 30C. Thesedata suggest that the dnaC gene product is not

TABLE 1. Synthesis of the high-molecular-weightand

normalforms ofqX174RFDNA at 41and30C in LD331 andH502a

%HMWb Rate at 41C/rate at 30 C

Expt-41 C 30 C HMW Normal Totalc

A, LD331 43 13 1.5 0.29 0.45

B, LD331 33 15 0.94 0.33 0.46

C, H502 12 16 1.2 1.7 1.6

aCellswere infected at 30C in 30

ltg

of

chloram-phenicol perml. Half ofeachculture was shifted to

41C at 45minafter infection.Equal portions of each

culture were pulse-labeled with [3H]thymidinefor 2

min(LD331)and 5 min (H502) at 20min(A) and (C)

and 30min (B) afterthetemperature shift. The rate

ofDNAsynthesiswas assumed to be proportional to

thecountsper minuteincorporated during the pulse.

bHMW, High molecular weight.

cTotal=HMWplus normal RF.

control where OX1743H-labeledRF DNA wasmixed with the 32P-labeled single-stranded DNA marker. Fractions were collected from the bottoms of the gradients. Arrows indicate the position of added

32P-labeledsingle-strandedOX174DNA marker.

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SYNTHESIS OF COMPLEX OX174

essential for the synthesis of the

high-molecu-lar-weight form of /X174 DNA, although it is

forthe synthesisofnormal /X174 RF DNA (12).

Thus, at 41 C the synthesis of this complex DNA continues in the dnaC mutant host, while

the synthesis of normal RF DNA is markedly

inhibited. This results in an increase in the

percentage of high-molecular-weight DNA at 41 C.

Function of the complex forms of

4X174

DNA. We asked whether the complex forms of

iX174 double-stranded DNA were

intermedi-ates in normal RF replication or dead-end products of abnormal DNA synthesis. The

in-tracellular phage DNA in LD331 was

pulse-labeled at 41 C and chased at 30 and 41 C.

Fast-sedimentinghigh-molecular-weight kX174 DNA was again detected in the lysate ofthe

pulse-labeled culture (Fig. 3A). This complex

DNA was not chased into normal RF DNA at

either 30(Fig. 3B) or 41 C (Fig. 3C).

kX174

RF

replicationresumes at a near normal rate upon

shifting down to 30 C under these conditions

(unpublished observation). These datasuggest

thatthecomplexform of kX174DNA is not an

intermediate in normal RF replication, but

rather a dead-end product.

Synthesis of the complex form of

OX174

DNA in a dnaE mutant. In the temperature-sensitive dnaEmutantLD301fast-sedimenting

kX174

DNAwas

synthesized

at30 C butnot at

41 C (Fig. 4). ThissuggeststhatDNA

polymer-ase III-catalyzed chain elongation is necessary

for the synthesis of the high-molecular-weight

qX174 DNA.

Structure of the complex form of

OX174

DNA. All of our data relevanttothestructure ofcomplex form of

kX174

DNA suggest that it consists of the kinds of oligomeric forms of

OX174

monomeric RF DNAobserved byothers in infections ofnormal host cells(1, 11, 15, 16).

When this DNA from sucrose

gradients

was

dialyzed, concentratedby alcohol precipitation,

and resedimented, we observed a partial

con-version to slower-sedimenting species. About

one-third still sedimented at the original rate

(approximately

30S),

while theremainder

sedi-mented at 14 to30S(datanot

shown).

supercoiled

circular dimers

of

OX174

RF DNA sediment at 29S, relaxed

circulardimers at21S,andhigherorderrelaxed

I0

x gO

0

10

20

30 FRACTION NUMBER

Fig. 1. At 40 min after infection 10-ml portions of the culture wereshifted to 41 C. At20minafter the

shift 100

ACi

of

[(H]thymidine

were added to each. Five minutes later one portion was rapidly chilled (A), while the other two were filtered. The latter

two were then washed with 10 ml of medium

con-taining 200jAg of thymine, 2 mgof thymidine, and

30 lAg of chloramphenicol per ml. The cells were

resuspended in 10 ml of the same medium and incubated 30 min at 30(B) and41 C(C). The cells

werecollected, washed,lysed,digested with protease, heated to 56 C for 20 min, and sedirrtented as

described inFig. 1.Arrows indicate thepositions of

added marker 32P-labeled 4X174 single-stranded

DNA. FIG. 3. Zone sedimentation of phage DNA

ex-tracted from 4,X174 am3-infected LD331

pulse-la-beled and chased during RF replication. The

con-ditionsfor culturingthecells,mitomycinC treatment, and infection were identical to those described in

1975

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1

2

D

6

4

2

0

10 20 20

FRACTION NUMBER

FIG. 4. Zone sedimentation of phage DNA

ex-tracted from OX174 am3-infected LD301

pulse-la-beled during RF replication. The conditions were

identical to those described in Fig. 1. After the mitomycin C treatment the cellswere suspended in

I volume ofTPGA mediumsupplemented with 0.01 ,ug of thymineperml. Phagewereaddedata

multi-plicity of 10 and chloramphenicol at 30 gg per ml.

After45 minat30 Chalf ofthe culturewasshifted

to 41 C. At 20 and 45 min after the shift 10-ml

portions of the 30 and 41 C cultures were

pulse-labeled for 2 min with [3H]thymidine, 10 uCi per

ml. A, 20 min, 30 C; B, 20 min, 41 C; C, 45 min, 30C;D,45min,41C.

oligomers somewhat faster than 21S (11). Su-percoiled and relaxed monomeric RF DNA molecules sediment at21and 16S, respectively. Thus, before these manipulations the complex form of

OX174

DNA observed in the dnaC

mutant host sedimented at a rate expected of

supercoiled dimeric and higher oligomeric forms ofRF DNA. After these manipulationsa hetero-geneous population of species sedimenting at

the ratesexpected of relaxed forms and

break-downproducts of these complex DNA molecules

was observed.

Zone sedimentation of the dialyzed,

concen-trated DNA from neutral pH sucrosegradients

in alkaline pH sucrose gradients showed three majorbands (Fig. 5). The DNA in the middle

band sedimented at the rate of denatured

monomeric RFI DNA. The DNA in the band

closesttothe bottomofthegradient sedimented

at the rate expected of denatured oligomeric

RFI DNA. The DNA in theslowest-sedimenting

band consisted of denatured single strands

derived from relaxed double-stranded DNA

molecules. When sedimented for longer times

(data not shown), the band was shown to be

more heterogeneous than that of an added

single-stranded circularDNAmarker. The data

suggested the presence of monomeric circular

and linear DNA strands, as well as oligomeric

single-strands.

When other preparations ofDNAfrom

neu-tral pH sucrose gradientswere spun to equilib-rium in CsCl gradients containing ethidium

bromide (100 ,ug/ml), about20% bandedat the

position ofsupercoiled DNA.

When dialyzed, concentrated DNA from

neu-tral pHsucrosegradientswasspread and

exam-8

2

6-0. 4

0

10

30

50 FRACTION NUMBER

FIG. 5. Zonesedimentation of the

high-molecular-weight kX174DNA in an alkaline sucrose gradient. LD331 was treated as described for sample B in Fig. 1. At 5 min after the shift to 41 C 200MCi of

['H]thymidine were added. Thirty minutes later the infected cells were collected and treated as described

inFig. 1. The DNA from the fast-sedimenting band

was dialyzed and concentrated by alcohol precipita-tion. This DNA sample was spun in an alkaline pH

sucrosegradient for 3.5hat 38,000 rpm in a Beckman

SW40 rotor at 5 C. Total radioactivity was measured

in each fraction collected from the bottom of the gradient.

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16, 1975 SYNTHESIS OF COMPLEXOX174DNA

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FIG. 6. Electronmicrographs of

OX1

74DNAfromLD331.A,Continuous tetramer;B,continuoustrimer;C,

continuousdimer; D,trimer,probablycatenated;F,monomers.The barrepresents0.51gm.

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ined by electron microscopy, monomer and complex forms of

OX174

DNA were observed. The complex formsincludedtetramers, trimers,

anddimers, some continuous, someapparently

catenated. Someexamplesareshown inFig.6.

These data suggest no unique structure for

the complex qX174DNAobservedinthednaC mutant host. The data indicate that these complex DNA molecules are similar in structure to at leastsome of those found by other inves-tigators in

OX174

infections of normal cells.

DISCUSSION

High-molecular-weight double-stranded forms of

OX174

DNA were synthesized during

theperiodofRFreplication (stage

II)

atboth 30

and 41C in a temperature-sensitive dnaC

mu-tant host. dnaC mutants are defective in the

initiation ofDNAsynthesis (2, 17). At41Cthe rate of

OX174

RF DNAsynthesisinthismutant hostwas 30% ofthat at 30C (Table 1). The

high-molecular-weightDNA wassynthesizedat least

asfastat 41 C asat30 C. Thissuggests that the formation of these complex DNA structures is

not dependent upon the activity of the dnaC

initiation protein.

Our data suggest that this complex DNA is

the productofDNAsynthesis, rather than DNA

recombination.Itsformation required the

activ-ity of the dnaE protein, DNA polymerase III (Fig. 4). This enzyme is essential for normal

OX174

RF replication (4). Also, more

radioac-tively labeled high-molecular-weight

OX174

DNA was made at 41 C during a short pulse than at 30C, while less radioactively labeled RF DNA was made (Fig. 1, Table 1). If the

radioactivity labeled complex DNA were a

product of recombination between normal RF DNA molecules synthesized during the period

ofthepulse, the rate of its formationshould

de-crease at 41C.

The high-molecular-weight 4X174 DNA was

not chased into

kX174

RF I and RF II DNA

molecules, the products of normal RF

replica-tion, at 30 or 41 C (Fig. 3). This indicates that this complex DNA is a dead-end product of abnormal DNA synthesis, not an intermediate in normalRF replication.

High-molecular-weight forms of

OX174

dou-ble-stranded DNA have been observed

previ-ously in normal infections, and in infections where RF replication was inhibited by phage mutations and drugs (1, 11, 15, 16). Both catenatedandcontinuous circles wereobserved. Thesestructuresapparently canarise from both DNA replication and DNA recombination.

Closed circularcomplex DNA structures

repre-sent 2 to 4% of the supercoiled RF DNA

synthesized under these conditions (11). Those

complex

DNA molecules stable to

repeated

centrifugation represent about 4% of the total

double-stranded RF DNA in the cell

(1).

In our experiments the complexDNA

mole-cules represented approximately 15% of the total

OX174

RF DNA synthesized at 30 C.

Twenty to fifty percent of this complex DNA

was completely closed circular, as seen by

analysisin alkalinepHsucrosegradientsand in

CsCl-ethidium bromide gradients. About

one-third still sedimented at 30S at neutral pH after the initial purification on sucrose

gradi-ents.The levels ofcomplexkX174 DNA thatwe

observed in infections at 30 C are therefore

comparabletothose observed inprevious

inves-tigations, the difference being that we did not

select only the completely closed circular DNA fraction or only the fraction stable torepeated centrifugation.

Thus the kinds of DNA molecules that accu-mulate in thednaC mutant host at the nonper-missive temperaturehave also been observed in this host at the permissive temperature and in normal host cells. Thesehigh-molecular-weight

OX174

DNA molecules apparently result from abnormal initiation of RF DNAsynthesissince theycontinue to be synthesized at a normalrate

under conditions where the initiation ofnormal RF replication is inhibited. The abnormal initi-ationpathway is usedinfrequentlyin normal in-fections since only a small amount of this complex DNA is synthesized. This pathway predominates only when the normal one is inhibited, leading to an accumulation of

com-plex qX174DNA.

ACKNOWLEDGMENTS

These results are drawn from athesissubmitted byE. G. K. to Northwestern Universityin partialfulfillment ofthe requirementforthe Ph.D. degree. We thank R. Ryanforhis helpwith theelectron microscopy, andH. Swift for theuse of hiselectron microscope facilities.

This work was supported by a Public Health Service research grant (AI-9882) and research careerdevelopment award(AI-70,632) to L. B. D. fromthe National Instituteof AllergyandInfectiousDiseases.

LITERATURE CITED

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tempera-ture-sensitive synthesis ofDNA. Mol. Gen. Genet. 109:107-122.

3. Davis,R.W.,M.Simon, andN.Davidson. 1971. Electron microscope heteroduplex methodsformapping regions ofbasesequencehomologyinnucleic acids,p.413-428. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. XXI. Academic Press Inc., New York.

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SYNTHESIS OF COMPLEX

OX174

DNA 419

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

5. Gefter, M. L., Y. Hirota, T. Kornberg, J. A. Wechsler, andC. Barnoux. 1971.Analysisof DNApolymerasesII and m in mutants ofEscherichia colithermosensitive

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6. Godson, G.N.,and D. Vapnek. 1973. A simple method of

preparing large amounts of OX174 RF supercoiled DNA. Biochim.Biophys.Acta 299:516-520.

7. Goebel, W. 1970. Studies on extrachromosomal DNA.

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temperature-sensitive mutants of Escherichia coli de-fective in DNA replication. Eur. J. Biochem. 15:311-320.

8. Goebel, W. 1974. Studies on the initiation of plasmid DNA replication. Eur. J. Biochem. 41:51-62.

9. Goebel, W., and D. R. Helinski. 1968. Generation of

highermultiple circular DNA forms inbacteria.Proc.

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10. Goebel, W., and J. Kraft. 1974. Complex col El DNA in Escherichia coli and Proteus mirabilis. Mol. Gen.

Genet. 129:149-166.

11. Gordon, C. M., M. G. Rush, and A. C.Warner. 1970.

Complexreplicative form molecules ofbacteriophage

*X174and S13su105.J.Mol. Biol. 47:495-503.

12. Kranias, E. G., and L. B. Dumas. 1974. Replication of bacteriophageOX174DNA in atemperature-sensitive dnaC mutant of Escherichia coli C. J. Virol. 13: 146-154.

13. Muller-Wecker, H., K. Geider, and H. Hoffmann-Berl-ing. 1972. DNA synthesis in nucleotide-permeable Escherichia coli cells. IV. Mode of1X174 replicative form DNAsynthesis and the templateinvolved. J. Mol. Biol. 69:319-331.

14. Nusslein, V., B. Otto, F. Bonhoeffer, and H. Schaller. 1971.Function of DNA polymerase III in DNA replica-tion. Nature(London) 234:285-286.

15. Rush, M. G., and R. C. Warner. 1968. Multiple length rings ofOX174and S13 replicative forms.III. A possible intermediate in recombination. J. Biol. Chem. 243: 4821-4826.

16. Rush, M. G., A. K. Kleinschmidt, W. Hellman, and R. C. Warner. 1967. Multiple length rings in preparation of

4X174replicativeform. Proc. Natl. Acad. Sci. U.S.A.

58:1676-1683.

17. Schulbach, W. H., J. D. Whitmer, and C. I. Davern. 1973.

Geneticcontrol of DNA initiation in Escherichia coli.

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18. Sinsheimer, R. L., B. Starman, C. Nagler, and S. Guthrie. 1962. The process of infection with bacterio-phageOX174.I.Evidence for areplicative form. J. Mol. Biol. 4:142-160.

VOL. 16,1975