JOURNAL OF VIROLOGY, June 1973, p. 848-855Copyright i 1973 American Society for Microbiology

Vol. 11, No. 6Printed in U.SA.

Replication of Bacteriophage qX174 DNA in a

Temperature-Sensitive dnaE Mutant ofEscherichia coli C

LAWRENCE B. DUMAS AND CHRISTINE A. MILLER

Department of Biological Sciences, and Program in Biochemistry and Molecular Biology, NorthwesternUniversity, Evanston, Illinois 60201

Received for Publication 17 January 1973

Bacteriophage 4X174 cannot grow in a temperature-sensitive dnaE (DNApolymerase III) mutant of Escherichia coli C at the nonpermissive temperature.The inability to grow is the result of inhibition of virus DNA synthesis. Thesynthesis of the parental replicative form is unaffected, but the replication of thereplicative form and the synthesis, of the single-stranded virus DNA are

inhibited.

Several DNA synthesis and DNA polymerasemutants of Escherichia coli have recently beenisolated. Mutants defective in genes polA andpolB, which code for DNA polymerases I and II,respectively, are able to replicate (2, 7). Thus,neither of these polymerases is essential forbacterial chromosome replication. Conditionallethal mutants in other loci have been isolatedand mapped (19). Mutants at one of these loci,dnaE, synthesize defective DNA polymerase IIImolecules (6, 14). This enzyme is required forreplication of the bacterial chromosome.

In examining the roles of the products of theseDNA synthesis loci in DNA replication, it isdesirable to follow the replication of a smallDNA molecule whose replicting intermediatescan be isolated intact. The bacteriophage4X174 DNA molecule and its replicating formscan easily be isolated and characterized. Thisvirus carries a limited amount of genetic infor-mation. It codes for no known DNA polymerase.Since it must use the E. coli DNA synthesissystem to replicate its DNA, it should be auseful probe in this examination.DNA polymerases I and II are not essential

for OX174 DNA replication (2). We have iso-lated a temperature-sensitive dnaE mutant ofE. coli C, the normal 4X174 host strain. Wereport here the effects of this mutation on thereplication of the DNA of this virus in vivo.

MATERIALS AND METHODSBacteria and phage strains. LD301 (uvrA-,

thyA-, endI-, dnaE") is a nitrosoguanidine-induced,

temperature-sensitive mutant of H502 (uvrA -, thyA -,endIl) isolated in our laboratory. The temperature-sensitive locus cotransduces with tonA at a frequencyof 60%, thus identifying this marker as dnaE (19). Themutant bacteria grow well at 30 C but cannot formvisible colonies on agar medium at 41 C. The details ofthe isolation of this mutant and its characterizationwill be published elsewhere.

Strain C is the standard host for 4X174 used here toprepare all phage stocks. HF4714 is an amber sup-pressor 4X174 host strain. OX174am3 is an ambermutant in gene E (lysis defective) and is the phageused in all experiments described in this report.

E. coli C, HF4714, and H502, and phage OX174am3were obtained from R. L. Sinsheimer.Media and buffers. TPGA medium is TPG me-

dium (17) with 1.0 g of KH2PO4 and 10 g of CasaminoAcids per liter. Starvation buffer has been described(3).Growth and purification of 4X174. Nonradioac-

tive phage were prepared as previously described (4).32P-labeled phage were grown as described by Sin-sheimer et al. (17), and purified as described byFrancke and Ray (5). The specific activity of thepurified phage was approximately 10-4 count per minper plaque-forming unit.

Cell lysis and Pronase digestion of the lysates.32P-phage infected cell samples were collected bycentrifugation, washed three times in 0.05 M sodiumtetraborate, 0.006M EDTA, pH 9, to elute radioactivephage particles still attached to the cell surface (12),and finally resuspended in 0.5 ml of the same buffer.Lysozyme was added to 0.2 mg/ml, and the suspen-sion was incubated at 0 C for at least 1 h. Incubationwas continued for several more hours at 0 C after theaddition of 1% Sarkosyl. After addition of 2 mg ofself-digested Pronase per ml the suspension wasincubated for 8 h at 37 C.

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4X174 DNA REPLICATION IN dnaE MUTANT

Cell samples from cultures infected with nonradi-oactive phage were washed and lysed in 0.05 M Tris,0.003 M EDTA, pH 8.1. All other conditions were asdescribed above.

Sucrose density gradient analysis. Lysed, Pro-nase-digested samples of phage-infected cells werelayered onto 38-ml linear gradients of 5 to 20% sucrosein 0.05 M potassium phosphate, 2 mM EDTA, 1 MNaCl, pH 7. These gradients were centrifuged for 16 hat 25,000 rpm at 4 C in a Spinco SW27 rotor. Fractionsof approximately 0.7 ml were collected from thebottoms of the tubes by using the perforator describedby Noll (13) (MICO, P.O. Box 1652, Evanston, Ill.).Portions of each fraction were diluted with water andmixed with 3 ml of scintillation fluid in 1-dram glassshell vials. The radioactivity in each vial was deter-mined in a liquid scintillation spectrometer. 5H radi-oactivity was corrected for 32p overlap. The scintilla-tion fluid consisted of 4 g of 2,5-diphenyloxazole(PPO) per liter in toluene-Triton X-100 (2:1).Measurement of acid-insoluble radioactivity.

Portions (0.1 ml) of cell cultures were transferred into1 ml of cold 5% trichloroacetic acid. A 200-,gg amountof highly polymerized calf thymus DNA was added as

carrier. The precipitates were collected on WhatmanGF/A glass fiber filters and washed three times withcold 5% trichloroacetic acid and finally with metha-nol. The air-dried filters were placed in 3 ml ofscintillation fluid (4 g of PPO per liter in toluene).Radioactivity was measured in a liquid scintillationspectrometer.Measurement of intracellular plaque-forming

units. Samples (0.1 ml) of the phage-infected cultureswere transferred into 1.2 ml of lysis buffer (0.4 Msodium tetraborate, 8 mM EDTA, 0.8% Brij 58, 1.7mg/ml lysozyme, pH 9). The mixtures were incubatedovernight at 0 C. After freezing and thawing twice, thelysates were diluted with 0.05 M sodium tetraborateand assayed by the agar overlay technique using hoststrain HF4714 as indicator.

Chemicals. Thymidine-methyl-3H, 10 to 25 Ci/mmol, and thymine-methyl-3H, 12.5 Ci/mmol, werepurchased from Intemational Chemical and NuclearCorp. and Amersham/Searle. 32P-phosphoric acid,carrier-free, was purchased from New England Nu-clear Corp. Mitomycin C, chloramphenicol, egg whitelysozyme, and Pronase (protease type VI) were pur-chased from Sigma Chemical Co.

RESULTS

OX174 growth at 41 C. To test whether hostmutant LD301 would support OX174 infection,a liquid culture of bacteria grown at 30 C wasinfected with *X174am3. Portions of the in-fected culture were incubated at 30 C and 41 C.Figure 1 shows that progeny phage are notsynthesized at 41 C in this host. Mature phageare synthesized at 30 C. After 3 h of infectionthe yield is approximately 50 PFU per cell.Phage synthesis at 30 C in the parent host

strain H502 occurs at a more rapid rate than inmutant LD301, and the total yield after 3 h of

a 1fI - - _

8.0

0

0

7.0-

0

6.0 _ _

0 2 3TIME (hours)

FIG. 1. Growth of bX174am3 in mutant hostLD301 at 30 and 41 C. The bacteria were grown at 30C to a cell density of approximately 2 x 10' cells perml on TPGA medium supplemented with 10 ug ofthymine per ml. The cells were collected and resus-pended in 0.1 volume of starvation buffer. 4X174am3was added to a multiplicity of 3. After 15 min withoutaeration, 1 volume of growth medium was added.Portions of the culture were aerated at 30 C and 41 C.At 30 and 60 min after aeration was resumed (zerotime), portions of the 30 C culture were shifted to 41C. At the indicated times 0.1-ml portions were with-drawn into lysis buffer. Total PFU were then deter-mined. Symbols: 0, PFU per ml from a cultureincubated at 30 C; 0, PFU per ml from a cultureincubated at 41 C; 0, PFU per ml from a cultureshifted to 41 C at 30 min; A, PFU per ml from a

culture shifted to 41 C at 60 min. Arrows indicate thetimes of shifts from 30 to 41 C.

infection is several-fold higher. At 41 C phagesynthesis is faster than at 30 C (unpublishedobservations).

Figure 1 also shows that temperature shiftsfrom 30 to 41 C at 30 and 60 min after infectionresult in a slowly decreasing rate of phagesynthesis, all synthesis having terminated by 30to 60 min after the shift.These results indicate the dnaE gene product

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DUMAS AND MILLER

of the host bacterium is required for the synthe-sis of progeny phage.4X174 DNA synthesis at 41 C. The tempera-

ture-sensitive mutation in the host bacteriumLD301 specifically affects host DNA synthesis.The effect of this mutation on kX174 DNAsynthesis was examined in phage-infected cellsby measuring the incorporation of 3H-thymineinto phage DNA at 30 and 41 C.

Bacteria grown at 30 C were treated withmitomycin C prior to infection to specificallyinhibit host DNA synthesis (11). Infected cellswere then incubated at the two temperatures.Figure 2 shows that the level of radioactiveDNA synthesized at 41 C in phage-infected cellsis no higher than that in uninfected cells in-cubated at 30 C and 41 C. No detectable phageDNA synthesis occurs.When 30 C cultures of infected cells were

shifted to 41 C at 30 and 60 min after infection,the phage DNA synthesis rate slowly decreased,reaching zero by 30 to 40 min after the shift. Thetermination of net phage DNA synthesis wasfollowed by a period of apparent degradation ofphage DNA. Under similar experimental condi-tions, cultures of uninfected mutant cells, nottreated with mitomycin C, cease net DNAsynthesis 30 to 40 min after a shift from 30 to 41C (unpublished data).These data indicate that the product of the

host dnaE gene is required for 4X174 DNAsynthesis.

Parental replicative form synthesis at 41 C.The replication of OX174 DNA occurs in threedistinct stages (16). In stage one the single-stranded DNA carried by the virus is convertedto the double-stranded parental replicativeform (RF) in the host cell by polymerization of acomplementary strand on the virus strand tem-plate. During stage two these double-strandedreplicative forms serve as template for thesynthesis of approximately 20 progeny RF mole-cules. In stage three the progeny RF moleculesserve as template for the asymmetric synthesisof many copies of the single-stranded virusDNA. We have examined the effect of the dnaEmutation in host strain LD301 on each of thesethree stages of kX174 DNA replication.To test the affect of this mutation on parental

RF synthesis, mutant host cell cultures grownat 30 C and preincubated at either 30 or 41 Cwere infected with 32P-labeled 4X174am3. Theconversion of the single-stranded virus DNA toRF molecules was examined by zone sedimenta-tion of lysates of the cultures in sucrose gradi-ents. In high ionic strength buffer the single-stranded DNA and the two double-strandedforms, RFI and RFII (closed, circular supercoils

and open, nicked circles, respectively), sedi-ment at rates of 23s, 21s, and 16s, respectively(1).

Figure 3A shows the results of such an analy-sis of a lysate from mitomycin C-treated cellsharvested 15 min after infection at 30 C. The32P-label is seen in a single-stranded DNA bandcentered about fraction 23, an RFI band cen-tered about fraction 32, and an RFH bandcentered about fraction 36. 3H-labeled RFI andRFII bands are also present, representingprogeny RF molecules synthesized during the

o0)

I0

x%-

2- 3TIME (hours)

FIG. 2. Radioactive thymine uptake into phageDNA in kX174am3 infected LD301. Bacteria weregrown at 30 C in 10 ml of TPGA medium supple-mented with 5 ug of thymine per ml to a cell density ofapproximately 3 x 108 cells per ml. The cells werecollected, washed in starvation buffer, and resus-pended in 1 ml of the same. Mitomycin C was addedto 0.1 mg/ml. After 20 min in the dark at 30 C, thecells were collected and resuspended in 2 ml ofstarvation buffer. Phage were added to 1.3 ml of thissuspension at a multiplicity of 3. After 15 min at 30 Cwithout aeration, 3 ml of fresh TPGA medium supple-mented with 1 Mg of thymine per ml and 15 MCi of3H-thymine were added to 0.3-ml portions of theuninfected and infected cultures (zero time). Portionsof each were incubated at 30 C and 41 C. One each ofthe infected cultures incubated at 30 C was shifted to41 C after 30 min and 60 min of aeration at 30 C.Acid-insoluble radioactivity was measured in 0.1-mlportions. Symbols: 0, 30 C, infected; 0, 30 C,uninfected; A, 41 C, infected; A, 41 C, uninfected; 0,shifted to 41 C at 30 min; *, shifted to 41 C at 60 min.Arrows indicate the times of shifts from 30 to 41 C.

850 J. VIROL.

V1X174 DNA REPLICATION IN dnaE MUTANT

N'-

0x

%..

A

6

4

2^toI0X

a-N

6cm4 )

2

I10 20 30 40 50 10 20 30 40 50

FRACTION NUMBERFIG. 3. Zone sedimentation of parental phage DNVA from infected LD301. Cultures (50 ml) of bacteria were

grown and treated with mitomycin C as described in Fig. 2. Portions (25 ml) of the treated cells in TPGAmedium supplemented with 1 gg of thymine per ml were aerated for 15 min at 30 C and 41 C (A and B,respectively) prior to infection. In a separate experiment 25-ml portions were incubated for 30 min at 30 C and41 C (C and D, respectively) prior to infection. 32P-OX174am3 were added to each culture at a multiplicity of 10.The specific activity of the phage was approximately 1 x 10-4 32p count per min per PFU in A and B andapproximately 5 x 10-5 32p count per min per PFU in C and D. 3H-thymidine (0.1 mCi) was added at the timeof infection. Each culture was incubated for 5 min without aeration, 15 min with aeration, and rapidly chilled.The infected cells were collected, washed, lysed, and digested with Pronase. The lysates were sedimentedthrough linear sucrose gradients as described in Materials and Methods. Total radioactivity was determined in0.2-ml portions of each fraction diluted with 0.1 ml of water. Symbols: , 'H; __, Sp.

15-min infection period. The "P-labeled single-stranded DNA band is due, at least in part, toDNA released during the lysis and Pronasetreatments from phage particles still attachedto the cell surface. The relative size of this banddecreases with the number of borate-EDTAwashes prior to lysis.A comparison of Fig. 3B to 3A shows that a

15-min preincubation of the mutant host cul-ture at 41 C prior to infection followed by a15-min infection period at 41 C caused noreduction in the amount of 32P-labeled phageDNA converted to RF relative to that at 30 C. Infact, there is 2.3-fold more 3"P-DNA in the RFbands. In contrast, the amount of 3H-labeledprogeny RF made at 41 C is only 40% that at 30C.

Similarly, preincubation of the mutant hostculture at 41 C for 30 min prior to infectioncauses no reduction in the conversion of pa-rental virus DNA label to RF at 41 C (compareFig. 3D to 3C). The amount of 'H-labeledprogeny RF made at 41 C in this experiment isonly 30% that at 30 C.

We have extended the preincubations at 41 Cto 1 and 2 h and find no decrease in theefficiency of conversion of parental DNA toparental RF at 41 C compared to that at 30 C.The 3H label in RF DNA from these 41 Ccultures is barely detectable and represents atmost 5% of that from the 30 C cultures (unpub-lished data).These results show that the product of the

host dnaE gene is not required for the synthesisof kX174 parental RF.4X174 RF replication at 41 C. The results of

the experiments described in Fig. 3 indicatethat kX174 RF replication is inhibited duringthe first 15 min of infection at 41 C. To examinethis inhibition in more detail, we pulse-labeled$X174-infected LD301 cultures at 15 and 30min after a shift from 30 to 41 C and comparedthe rates of RF DNA synthesis at the twotemperatures. The cultures were treated withmitomycin C prior to infection and infected inthe presence of 30 ,g of chloramphenicol per ml.This concentration of chloramphenicol allowsRF replication to proceed for several hours and

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prevents single-stranded virus DNA synthesis(17).Lysates of these pulse-labeled cultures were

then sedimented through sucrose gradients.Figure 4A shows the sedimentation profile of thelysate of the 30 C culture, and Fig. 4B showsthat of the 41 C culture, pulse-labeled at 15 minafter the shift. The radioactive label in the RFregion in gradient B is only about 17% of that ingradient A.

In a separate experiment a portion of asimilarly infected culture was incubated for 30min after the shift to 41 C before pulse-labeling.Figure 4D shows that the amount of 3H label inRF DNA from the 41 C culture is again onlyabout one-fifth that from the 30 C culture (Fig.4C).When 4X174-infected cultures of LD301 were

pulse-labeled during the period of RF replica-tion immediately after the shift to 41 C, weobserved 2.4-fold more pulse-label in RF DNA

6 A

4

0

K

6

from lysates of 41 C cultures than from identicalcultures at 30 C (data not shown). The rate ofRF replication at 15 and 30 min after the shift istherefore approximately 7 to 8% of the initialrate at 41 C.The results of these experiments show that

the product of the host dnaE gene is required forthe replication of kX174 RF DNA.Single-stranded phage DNA synthesis at

41 C. A culture of LD301 was infected withkX174am3 and incubated at 30 C for 45 min. By

this time single-stranded virus DNA is beingsynthesized and encapsulated in mature phageparticles (see Fig. 1). Label incorporated intoRF molecules during short pulses at this stage isfound predominantly in the viral strand andchases into virus particles (10).

Half of the culture was then shifted to 41 C.At 10, 20, and 30 min after the shift, portions ofthe 30 C and 41 C cultures were pulse-labeledwith 3H-thymidine for 1 min. The labeled phage

10 20 30 40 50 10 20 30 40 50

FRACTION NUMBERFIG. 4. Zone sedimentation of phage DNA from X1 74-infected LD301 pulse-labeled during RF replication.

Bacteria cultures were grown and treated with mitomycin C as described in Fig. 2. Treated cells wereresuspended in TPGA medium supplemented with I .tg of thymine and 30 ,ug of chloramphenicol per ml. Phagewere added at a multiplicity of 3. The cultures were incubated at 30 C. At 20 min after addition of phage,one-half of the culture was shifted to 41 C. After 15 min 10 ml of the 30 C and the 41 C cultures werepulse-labeled for 1 min with 125 MCi of 8H-thymidine (A and B, respectively). In a separate experiment, similar10-ml cultures of 30 C and 41 C were pulse-labeled for 1 min at 30 min after the shift to 41 C. The labeledcultures were rapidly chilled by the addition of an equal volume of acetone chilled in a methanol-dry ice bath.The cells were collected, washed, lysed, and digested with Pronase. The Iysates were sedimented through linearsucrose gradients as described in Materials and Methods. Arrows indicate the peak positions of added'2P-labeled 4X174 virus DNA marker. Total radioactivity was determined in 0.4-ml portions of each fractiondiluted to 1 ml with water.

852 J.- VIROL.

VX174 DNA REPLICATION IN dnaE MUTANT

DNA from these cultures was then examined byzone sedimentation.

Figures 5A and 5B show the sedimentationprofiles of the DNA pulse-labeled at 30 C and 41C, respectively, at 10 min after the temperatureshift. Figures 5C and 5D represent similaranalyses of portions of the two cultures pulse-labeled at 20 min after the shift, and Fig. 5Eand 5F at 40 min. In all of the gradients the RFIIband is centered near fraction 30, and the freesingle-stranded virus DNA band position,marked by added 32P-DNA, is indicated by thearrow. Phage DNA not liberated from phageparticles during the Pronase digestion is foundon the dense cushion at the bottom of eachgradient.The amount of radioactive label incorporated

into the phage DNA species during the 30 Cpulses at 10, 20, and 40 min after the shift isapproximately the same. The rate of phageDNA synthesis at 30 C during this time period istherefore constant. The rate at 41 C, however,decreases. Comparison of Fig. 5B to 5A showsthat at 10 min after the shift phage DNAsynthesis is occurring at a 3.2-fold greater rateat 41 C than at 30 C. Ten minutes later the rateat 41 C has decreased to 54% of that at 30 C. at40 min after the shift the 41 C rate is only 26% ofthat at 30 C. The rate of virus DNA synthesis at

20 and 40 min after the shift is, therefore,approximately 17 and 8%, respectively, of thatat 10 min after the shift.Single-stranded virus DNA and phage parti-

cles are synthesized at 41 C, even after 40 min atthis temperature. But the rate of synthesis hasbeen reduced to only 8% of the initial rate at 41C.These results show that the product of the

host dnaE gene is required for the synthesis ofsingle-stranded 4X174 DNA.

DISCUSSION4X174 RF replication occurs on the cell

membrane (9), as does E. coli DNA replication(18). The replication of the E. coli chromosomerequires DNA polymerase II (6, 14). We haveshown here that RF replication is inhibited atthe nonpermissive temperature in a dnaE mu-tant. The rate of synthesis initially increasesafter shifts to 41 C, but is approximately 90%inhibited by 15 min after the shift. Thus DNApolymerase III is also required for kX174 RFreplication.The late synthesis of 4X174 single-stranded

DNA occurs in the cell cytoplasm (8). Thissynthesis is also inhibited in the dnaE mutanthost at 41 C. This reaction is approximately 80%inhibited after 20 min at the nonpermissive

0

4~~~~~~~

10 20 30 40 10 20 30 40 10 20 30 40FRACTION NUMBER

FIG. 5. ZonesedimentationofphageDNAfrom<qX174-infectedLD301pulse-labeledduringsingle-strandedDNA synthesis. A 60-ml culture of bacteria in TPGA plus thymine was incubated at 30 C until the cell densityreached approximately 5 x 108 cells per ml. The cells were collected, washed in starvation buffer, andresuspended in 5 ml of the same. Phage were added at a multiplicity of 3, and the suspension was incubated for15 min at 30 C without aeration. At zero time, 55 ml of TPGA medium plus 2 ,gg of thymine per ml were added.After 45 min of aeration at 30 C, one-half of the culture was shifted to 41 C. At 10, 20, and 40 min after thetemperature shift, 100 MCi of 3H-thymidine were added to 10 ml portions of the 30 C and 41 C cultures. After Imin each sample was rapidly chilled and processed as described in Fig. 4. The gradients in this experimentconsisted of 34 ml of buffered 5 to 20%o sucrose over a 3-ml cushion of saturated CsCI in buffered 60% sucrose. A,10 min at 30 C; B, 10 min at 41 C; C, 20 min at 30 C; D, 20 min at 41 C; E, 40 min at 30 C; F, 40 min at 41 C.Note that the ordinate in gradient B is different from all of the others.

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temperature. We conclude that DNA polym-erase III is also required for the synthesis ofOX174 single-stranded DNA.The synthesis of 4X174 parental RF is not

inhibited in this dnaE mutant. The conversionof parent phage DNA to the double-strandedreplicative forms occurs even after 1 to 2 h ofpreincubation of the culture at 41 C. Little or noprogeny RF replication occurs after such pro-longed preincubation. We conclude that DNApolymerase III is not required for parental RFsynthesis in this host.

Others have shown that kX174 can grow inmutants defective in DNA polymerases I and II(2, 7). Presumably parental RF synthesis occursin these mutants, indicating that neither ofthese polymerases is required in cells that havenormal DNA polymerase Ill activity.

Further evidence concerning the possibleroles of the three known DNA polymerases inparental RF synthesis comes from experimentsusing cell-free extracts. Extracts ofpolA- polB+dnaEts E. coli are defective in the conversion ofadded 4X174 virus DNA to the double-strandedform (15, 20). Extracts of polA- polB+ dnaE+cells are not defective. Thus, DNA polymeraseIII is required for the in vitro reaction in extractsdefective in DNA polymerase I activity. Ex-tracts of polA- polB - dnaE+ bacteria can cata-lyze the in vitro reaction (20). Thus, DNApolymerase II activity is neither required norsufficient.A possible explanation for the requirement of

DNA polymerase III activity for the conversionof 4X174 virus DNA to a double-strandedmolecule in these cell extracts, and no suchrequirement for parental RF synthesis in vivo inLD301, is that both DNA polymerases I and IIImight act as catalysts for these reactions. Theabsence of only one of these two polymeraseactivities would not prevent the conversion ofvirus DNA to double-stranded DNA. Since hostLD301 is defective in DNA polymerase III, butnot DNA polymerase I, parental RF synthesisoccurs. The cell extracts, defective in bothpolymerase activities, do not catalyze the con-version. This explanation can be tested in vivoin a polAl- dnaEt5 double mutant. If correct,parental RF synthesis should not occur at 41 C.

If the in vitro conversion of 4X174 DNA to adouble-stranded molecule is different from thesynthesis of parental RF in vivo, then we cannotexclude DNA polymerase II from a possible rolein vivo. We can only say that some DNApolymerase other than III can catalyze the invivo reaction in LD301. DNA polymerase IIIcannot be excluded from participating in nor-mal hosts. The DNA polymerase catalyzing

parental RF synthesis in LD301 could be polym-erase I or II, or a new host cell enzyme.

ACKNOWLEDGMENTSThis work was supported in part by Public Health Service

research grant number 9882 from the National Institute ofAllergy and Infectious Diseases, research grant numberGB22867 from the National Science Foundation, and researchgrant number 70-21 from the American Cancer Society,ilinois Division, Inc.

LITERATURE CITED1. Burton, A., and R. L. Sinsheimer. 1965. The process of

infection with bacteriophage 4X174. VII. Ultracentrif-ugal analysis of the replicative form. J. Mol. Biol.14:327-347.

2. Campbell, J. L., L. Soll, and C. C. Richardson. 1972.Isolation and partial characterization of a mutant ofEscherichia coli deficient in DNA polymerase II. Proc.Nat. Acad. Sci. U.S.A. 69:2090-2094.

3. Denhardt, D. T., and R. L. Sinsheimer. 1965. The processof infection with bacteriophage 4X174. m. Phagematuration and lysis after synchronized infection. J.Mol. Biol. 12:641-646.

4. Dumas, L. B., G. Darby, and R. L. Sinsheimer. 1971. Thereplication of bacteriophage OX174 DNA in vitro.Temperature effects on repair synthesis and displace-ment synthesis. Biochim. Biophys. Acta 228:407-422.

5. Francke, B., and D. S. Ray. 1971. Fate of parental 4X174DNA upon infection of starved thymine-requiring cells.Virology 44:168-187.

6. Gefter, M. L., Y. Hirota, T. Kornberg, J. A. Wechsler,and C. Bamoux. 1971. Analysis ofDNA polymerases IIand III in mutants of Escherichia coli thermosensitivefor DNA synthesis. Proc. Nat. Acad. Sci. U.S.A.68:3150-3153.

7. Hirota, Y., M. Gefter, and L. Mindich. 1972. A mutant ofEscherichia coli defective in DNA polymerase II ac-tivity. Proc. Nat. Acad. Sci. U.S.A. 69:3238-3242.

8. Knippers, R., A. Razin, R. Davis, and R. L. Sinsheimer.1969. The process of infection with bacteriophage4OX174. XXIX. In vivo studies on the synthesis of thesingle-stranded DNA of progeny fX174 bacteriophage.J. Mol. Biol. 45:237-263.

9. Knippers, R., and R. L. Sinsheimer. 1968. The process ofinfection with bacteriophage OX174. XX. Attachmentof the parental DNA of bacteriophage 4X174 to afast-sedimenting cell component. J. Mol. Biol.34:17-29.

10. Komano, T., R. Knippers, and R. L. Sinsheimer. 1968.The process of infection with bacteriophage 4X174.XXII. Synthesis of progeny single-stranded DNA.Proc. Nat. Acad. Sci. U.S.A. 59:911-916.

11. Lindquist, B. H., and R. L. Sinsheimer. 1967. The processof infection with bacteriophage OX174. XV. Bacterio-phage DNA synthesis in abortive infections with a setof conditional lethal mutants. J. Mol. Biol. 30:69-80.

12. Newbold, J. E., and R. L. Sinsheimer. 1969. The processof infection with bacteriophage OX174. XXXII. Earlysteps in the infection process: attachment, eclipse, andDNA penetration. J. Mol. Biol. 49:49-66.

13. Noll, H. 1969. Polysomes: analysis of structure andfunction, p. 101-179. In J. Sargent, and P. N. Campbell(ed.), Techniques in protein biosynthesis, vol. 2. Aca-demic Press Inc., London.

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

15. Schekman, R., W. Wickner, 0. Westergaard, D. Brutlag,K. Geider, L. L. Bertsch, and A. Kornberg. 1972.Initiation of DNA synthesis: synthesis of OX174 rep-

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licative form requires RNA synthesis resistant torifampicin. Proc. Nat. Acad. Sci. U.S.A. 69:2691-2695.

16. Sinsheimer, R. L., R. Knippers, and T. Komano. 1968.Stages in the replication of bacteriophage *X174 invivo. Cold Spring Harbor Symp. Quant. Biol.33:443-447.

17. Sinsheimer, R. L., B. Starman, C. Nagler, and S.Guthrie. 1962. The process of infection with bacterio-phage OX174. I. Evidence for a replicative form. J. Mol.Biol. 4:142-160.

18. Smith, D. W., and P. C. Hanawalt. 1967. Properties of thegrowing point region of the bacterial chromosome.Biochim. Biophys. Acta 149:519-531.

19. Wechler, J. A., and J. D. Gross. 1971. Escherichia colimutants temperature-sensitive for DNA synthesis.Mol. Gen. Genet. 113:273-284.

20. Wickner, R. B., M. Wright, S. Wickner, and J. Hurwitz.1972. Conversion of OX174 and fd single-stranded DNAto replicative forms in extracts of Escherichia coli.Proc. Nat. Acad. Sci. U.S.A. 69:3233-3237.

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