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Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

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JouRNALoF VImoLoGY, May 1976, p. 426-435 Copyright X 1976 AmericanSocietyforMicrobiology

Vol. 18, No.2

Printed inU.SA.

Bacteriophage

4X174

Single-Stranded

Viral DNA

Synthesis

in

Temperature-Sensitive dnaB and dnaC

Mutants of

Escherichia coli

LAWRENCE B. DUMAS* AND CHRISTINE A. MILLER

Department of Biochemistry and MolecularBiology,Northwestern UniversityEvanston,Illinois60201 Received for publication 8December 1975

We asked if 4X174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature indnaB and dnaC

temperature-sensitive host

mu-tants.

The

rates of

single-stranded

DNA

synthesis

were measured after the

removal of chloramphenicol thathad been

added

at varioustimesafter infection

to

specifically

stop this stage of 4X174 DNA synthesis. Reinitiation was not

defective ineithermutanthost. Our data

suggested

that the reinitiation of the

single-stranded

stageof 4X174 DNA

synthesis

inthese

experiments

was

analo-gous tothe normal initiation of this stage of 4X174 DNAsynthesis in infections without chloramphenicol. Assuming thisto be the case, we

conclude

thatthe host cell dnaB and dnaC proteins are not essential for the normalinitiationof

the single-stranded synthesis stage of

4X174

DNA synthesis. In related experi-ments weobserved that in the dnaC mutant host at the permissive temperature,

OX174

replicative form DNAsynthesiscontinuedatitsinitialrate even

during

the single-strandedDNAsynthesis stage. This indicates thatthesetwostagesof

4X174

DNAsynthesisarenotnecessarily mutually exclusive.

The Escherichia coli dnaC protein is

re-quired for the initiation ofcycles of chromosome replication in vivo (1, 10), whereas the dnaB protein isessential for continued DNA

synthe-sis once the primary initiation event has

oc-curred (4, 12). The dnaB and dnaC

proteins

participate inthe initiationof the

synthesis

of the complementary strand of

bacteriophage

OX174

on the

single-stranded (SS)

viral DNA templateinvitro (9, 13).

Both the dnaB and dnaC proteinsare

essen-tial for

4X174

DNA

synthesis

invivo(3,6). The synthesis of this phage DNA occurs in three stages (11).Neither proteinis

directly

involved

inthe continuationof

OX174

SS DNA

synthe-sis, the last of the three stages, once it has begun (3, 6). However, both of these host

pro-teins are essential forthe replication of

4X174

replicative form (RF)DNA (3, 6), the template for late SS DNAsynthesis. These twoproteins

arethereforeindirectly essential for

OX174

SS

DNAsynthesis.

We asked ifeither the dnaB or the dnaC

protein of E. coli is directly essential for the

initiation of

4X174

SSDNAsynthesis. We

pres-ent evidence here indicatingthat neither

pro-tein isrequired.

MATERIALS AND METHODS

Bacteria and phage strains. LD301 (uvrA-,

thyA-, endI-,dnaEis), LD311(uvrA-, thyA-, endI-,

dnaB's), and LD332 (uvrA-, thyA-, endL-, dnaCls)

aretemperature-sensitive mutantsof H502 (uvrA-, thyA-, endI-) isolated in our laboratory (2, 3, 6). Stocksof4X174am3 (geneE, lysis defective) were prepared using E. coli C as host.

Rate of X174 DNA synthesis measurements. A 200-ml culture of bacteria was grown at 30 C to a cell density of4 x 108cells per mlinTPGAmedium (2)

supplementedwith2,ugofthymine per ml. The cells

were collected and treated with mitomycin C as previously described (3). After suspension in 10 ml of TPGA medium plus thymine,4X174am3 was added

at amultiplicityofinfection of3.After 5 min at 30 C

the culture was diluted to 200 ml with the same medium (zero time). At various times after infection portions of this culturewereshifted to41C. In some experiments chloramphenicolwasadded at a final concentration of30

Ag

per mlatthe time of the shift. Thedrug was removed 45 min later by filtration at

41C. Atregular intervals 2-mlportions of the cul-tures were pulse labeled with 5 ,uCi of

[3Hlthymidine for 2 min. The pulses were

termi-nated by the addition of an equal volume of pre-chilled acetone. The cells werecollected by centrifu-gation and suspended in 1 ml of 50 mM sodium tetraborate-10 mM EDTA-200

.g

of lysozyme per ml (pH 9). After 20 min at room temperature KOH was added to 0.3 N. The mixture was incubated over-night at 30 C. Calf thymus DNA (200

jig)

and 10% trichloroacetic acid (2 ml) were added. The precipi-tates were collected and washed on glass-fiber fil-ters.Radioactivity was measured in a liquid scintil-lation spectrometer.

Sucrose density gradient analysis of qbX174 DNA. The bacteria were grown,mitomycin C treated, and

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pH 8, and suspended in 1 ml of the same buffer.

Lysozyme was added to 200 uig per ml, and the ri 16 _ | mixturewasincubated 20

min

at0 C. Sarkosylwas

addedto3%, and the mixture was incubatedover- N night at 0 C. Protease (2 mg/ml, Sigma type VI) was

added,

and the mixture was incubated for8 h at

37 C. The

samples

were then heated for 20 minat 12

-56 C.

32P-labeled

4X174

viral DNA marker was

added. The samples were then layered onto 36-ml

lineargradientsof 5to20%sucrosein50 mMpotas-o l

sium

phosphate-2

mM EDTA-1 M

NaCl,

pH

7.These |

werespun 16 h at 10 C at 24,000 rpm in aBeckman 0 :I tVV SW27 rotor. Fractions werecollected from the bot- ::d

tomsof the tubes into 1-dramglass shellvials. Ra- E i%

dioactivity was measured in a liquid scintillation L

aO..2

| /

spectrometer. W 0

RESULTS 4_ yA

Rateof4X174 DNAsynthesisinthe parent

and mutant hosts. We measured therates of 4

40 80 120 160 10__

10 TIME(min)

FIG. 2. Rates ofXX174 DNA synthesis at 30 and

41 CinhoststrainLD332(dnaCls). At0, 30, 60,and

A,

3 75 min after infection, portions of the culture were

T 8 ° ° %

shifted from

30to41 C.

Symbols: *,

rateat30

C;

0,

2 * 3% ~ rateat41 C,shiftedat0min;A,rateat41 C,shifted

N I at30

min;

,rate at41 C,shiftedat60 min;

V,

rate

X

' 5 1 at41 C,shiftedat75 min.

3%

bfi

6-

faX174

1 | t \

am3

lysiss

defective) DNA

synthesis

in

U I3 ° i host cellsat30C and after

shifting

to 41 C.The

.C

9e

8 %

\rates

were determined at various times after

% infection

by

measuring the amounts of

0 1lXt

ry \

[3H]thymidine

incorporated

into small

portions

40842- AdoftheAd *

mitomycin

C-treatedinfected cell culture

E I

b.

during2-minpulses. The results from a

OX174-infected culture of the nondefective

parental

w 0 I host strain H502 are shown in Fig. 1. At 30 C

,w # I "'"' the rate of

OX174

increased continuously for

t2 - /p.r > & and% Ad about 40 min after infection and then

slowly

d

% declined. Most of the DNA

synthesized

at 30C

t1/ X after

approximately

20 to 30 min

postinfection

V

,41 in this kind ofexperiment was SS DNA (see

below). Portions of the culture were shiftedto

0-- _j |,41Cat0, 15, 30, and45minafter infection.The

40 80 120 maximum rate of

OX174

SS DNA synthesis

TIME(min) observed at 41 C was at least as high as that at

30C,no matterwhenthe culturewasshiftedto

FIG. 1. Rates of(*Xl174DNA synthesis at 30 and the higher temperature.

41 Cintheparenthost strain H502. At0, 15,30and theThe results fromr e

rac

a

comparable experiment

p b e n

45 min after infection portions oftheculture

wereenwe

shifted from30 to 41 C.Symbols: 0,rate at30C; 0, using ma

X174-infected

temperature-sensitive

rateat 41C, shiftedat0min; A,rateat 41 C,shifted dnaCmutanthost cultureare shown inFig. 2.

at 15min; O,rate at 41 C,shiftedat30min;V,rate SS DNA synthesis began at approximately 30

at41 C, shiftedat 45min. min

postinfection

in this mutant at 30

C,

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428 DUMAS AND MILLER

o 2

E

0 8

01% %~~~~~~~~~

CLJ

4 %

40 80 120 160

TIME

(min)

FIG. 3. Rates of4X174 DNAsynthesis at 30 and 41 C in the parent host strain H502. At 10, 30, and 50 minafter infection, chloramphenicol (30 HgIml) was added to 50-ml portions of the culture. These same portionswerethenshiftedto 41C. At 55, 75, and 95min, respectively, the drug was removed by filtrationat 41 C. Symbols: V, rate at 30 C, no chloramphenicol added;0,rate at 41 C, shifted at 10min, chlorampheni-col removedat55min;0,rateat 30C after removal ofchloramphenicol at 55 min; A, rate at 41 C, shiftedat

30minm chloramphenicolremovedat75 min; A, rateat30C after removal ofchloramphenicol at 75 min; O,

rate at 41 C, shifted at 50

min,

chloramphenicol removed at 95 min; *, rate at 30C after removal of chloramphenicolat95 min.

mostof the DNA synthesizedat30 C after

ap-proximately 40 to 60minpostinfection was SS

DNA (seebelow). At 30 C the rateof SS DNA

synthesis increased continuously for about 2.5h

after infection. The slower, more prolonged

synthesiswasprobablydue tothemuchslower

rate of RF template DNA replication in the

mutanthost (6; see below). Shifts to 41 C, the

nonpermissive temperature for the dnaC

pro-teinactivity, inhibited SS DNA synthesis,

es-pecially when carried out early in infection.

Similar observations were made using the

dnaB mutanthost(3; seebelow).

Reinitiation of single-stranded DNA

syn-thesis. Thesensitivityof 4X174 DNAsynthesis

to early temperature shifts in the dnaB and

dnaCmutantscould besimply duetoinhibition

ofthe synthesis of RF DNA molecules (3, 6),

which actastemplates for SS DNA synthesis,

ortothe directinhibition of the initiation of the

SS DNAsynthesis stage, orboth. Neither the

dnaBnorthednaC mutationdirectly affects SS

synthesisonceit hasbegun (3, 6).

Wetested the possibility that the dnaB and

dnaC mutationsmight directly affectthe

initi-ation of SS synthesis. The experiments

re-quiredtheaddition of30

,.tg

ofchloramphenicol

per ml to the infected cell cultures at various

times after infection with simultaneous shifts

to 41 C. After thistreatment, SS DNA

synthe-sis already begun proceeds at a continuously decreasing rate asthe viral proteins, the

syn-thesis of which is inhibited by the chloram-phenicol, are used up (7). Eventually SS

syn-thesisceases(7).Innormal hostcells, RF

repli-cationcontinues

after

the additionof

chloram-phenicol (5, 7). Inthe dnaB and dnaC mutant

hosts RF replication is inhibited due to the

temperature shift (3, 6).Inourexperiments the

chloramphenicol was later removed by

filtra-tion at 41C. We then monitored the rate of

pX174

DNA synthesis to see if SS synthesis couldreinitiate at41 C.

Figure 3 shows the results of the control

ex-periment using the parent host strain.The

in-fected cellswereshiftedto 41Cat10,30,and50

minafterinfection,withsimultaneous addition

ofchloramphenicolto30

tmg/ml.

After 45 minat

41C the cultures were filtered. The cellswere

suspended in medium without

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SYNTHESIS OF SINGLE-STRANDED

OX174

DNA

429

10x0

WI

I

I

0~

0

16-12

8

-4

20

40

60

20

40

60

FRACTION NUMBER

FIG. 4. Zonesedimentation of pulse-labeled intracellular 4X174 DNA extracted from parent host strain H502. At 25 min after infection a portion of the culture was pulse-labeled at 30 C (A). At 30 min after infection the remainder of the culture was shifted to 41 C, and chloramphenicol was added at 30pg/ml.At 70min a portionof the culture was pulse-labeledat 41 C (B). At75 minafter infection the remainder ofthe culture was

filteredat 41C. Half was further incubated at41 C, and half was further incubated at 30 C. At 110 min a

portion of the 41 C culture was pulse-labeled (C). At 140 minaportion of the 30 C culture was pulse-labeled (D). The arrows represent the positions of the added32P-labeled

0X1

74 virusDNAmarker.

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430 DUMAS AND MILLER

o

0~~~~

o

.s

4-'~~~~~~~~~~~

,

9%2,t

E

/

\

R

2 %%~~~~~~~~~~I~~

I % ~~~~.Ie ~ %

C

~~~40

80 120 160 TIME

(min)

FIG. 5. RatesofXX174DNAsynthesisat30and41 CinhoststrainLD311(dnaB's).At10, 30, and50 min

after infection, chloramphenicol wasaddedtoportionsofthecultureatafinalconcentrationof 30 pg/ml. These same portionsweresimultaneouslyshiftedto 41 C. At45minafter the shifts, the chloramphenicolwas

removed by filtration at41 C.Symbols: *,rate at30C,nochloramphenicol added;0,rate at41 C, shiftedat

10min,chloramphenicolremovedat55min; A,rateat41 C, shiftedat30min,chloramphenicol removedat

75min; O,rate at 41C,shiftedat50min, chloramphenicolremovedat95min.

col.InallcasesSSsynthesis reinitiatedat41 C.

SS synthesis also reinitiated at 30 C, but the

samemaximumrates were notachievedduring

thetimeobserved. When chloramphenicol was

added back to the medium after filtration, SS synthesis failedto reinitiate

(data

not

shown).

Figure4shows thezonesedimentation

anal-ysesof the

OX174

DNA

synthesized

inanother culture at various times during this kind of experiment. Three distinct viral DNA species could be seen inthese

analyses:

SS DNA and

the two

double-stranded

forms RFI and RFII

(closed,

circular

supercoils

and open, relaxed

circles, respectively). These three species

sedi-ment at ratesof27, 21, and 16S,

respectively.

The radioactivity at the top ofeach gradient

represents unincorporated [3H]thymidine.

Im-mediatelybefore the temperature shiftandthe

addition of chloramphenicol, pulse-label was

found in SS DNA and in RF DNA (Fig. 4A).

Justbefore theremovalof thechloramphenicol,

pulse-label was found in RF DNA (Fig. 4B).

But little or no label was found in SS DNA.

After the removal of thedrug pulse-label

incor-porated at 41 and 30C was found

predomi-nantly in SS DNA (Fig. 4C and D,

respec-tively). These data confirm the results

ex-pected: addition of

chloramphenicol

causedthe

eventual cessation of SS DNA synthesis, whereas removal ofthe drug allowed SS

syn-thesisto reinitiate.

Figures 5 and 6show the results of

compara-ble experiments using the dnaB mutant host. Figure5shows that the simultaneous shiftto 41

C andaddition of chloramphenicol resulted in nearcomplete inhibition of DNAsynthesis.

Re-moval of the

chloramphenicol

allowed reinitia-tionof

4X174

SS DNAsynthesisat 41 C.Figure 6A shows that the 25 min after infection in

another.culture

at30 C,

immediately

priortoa

temperature shift, SS DNAwas being

synthe-sized. Immediately prior to the removal of chloramphenicolat41 CnodetectableSSDNA

synthesis was occurring (Fig. 6B). After

re-moval of thechloramphenicol,SS DNA

synthe-siswasobservedat41and30C (Fig.6CandD, respectively).These datashowthatreinitiation

of SS DNAsynthesisoccursat 41 Cinthe dnaB

mutantatapproximatelythesameefficiency as

intheparent host strain. The dnaB protein is

thereforenotessentialfor the reinitiationof SS DNAsynthesis.

Figures 7and 8 show the results of

compara-bleexperiments using the dnaC mutant host.

Again removal ofthe chloramphenicolallowed reinitiation of SS DNA synthesis at 41 C (Fig. J. VIROL.

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16

12

to

4

%

_

I

I

I

2

C

D

16

-12

-

h

8

4-20

40

60

20

FRACTION NUMBER

FIG. 6. Zone sedimentationofpulse-labeledintracellularMAXl74DNA extractedfromLD311 (dnaB5).The

experimentwascarried outexactlyasdescribedin thelegendtoFig.4.

40

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432 DUMAS AND MILLER

24p

9%~~~~~~~~~~~~ 0 0

16

18

~~~~~~~~~it

I

0

1

060 120 180

TIME

(min)

FIG. 7. Ratesof

qX1

74DNAsynthesisat30and

41 Cin host strain LD332 (dnaC"s). At60,80,and 100 minafter infection, chloramphenicolwasadded

toportionsofthe cultureatafinalconcentrationof30

pg/ml. These same portions were simultaneously

shiftedto41 C. At 45minafter theshifts thedrug

wasremovedby filtrationat41 C. Portions(2 ml) of

the cultureswerepulse-labeledwith 10 juCiof

thymi-dine,twiceasmuchasinthe analogousexperiments

described above. Symbols:0,rate at30C,no

chlor-amphenicol added;0,rate at41 C, shiftedat60min,

chloramphenicol removed at 105 min; A, rate at 41 C,shiftedat80min,chloramphenicolremovedat 125min; E,rate at41 C,shiftedat100minm

chlor-amphenicolremovedat145min.

7). Immediately prior to the removal of the

drug, SS DNA synthesis wasnotdetectable in

another culture (Fig. 8B), whereas after its

re-movalSS DNAwassynthesizedatarelatively

rapid rate at41 C (Fig. 80). At 30 C SS DNA

synthesis was very slow (Fig. 8D). Since the

reinitiationof SS DNA synthesisoccursat41 C

as efficiently as in the parenthost strain, we

conclude thatthe dnaC proteinis notessential

for thereinitiationof SS DNAsynthesis.

Previous investigations showed that

OXi74

SS DNAsynthesiswasinhibitedat41 C latein

infectioninatemperature-sensitive dnaE

mu-tant host (2) and in adnaG mutant host (8).

When theabilitytoresumeSS DNA synthesis

at41 C was examined inthe dnaE mutantin

experiments comparable to those described

above, we found that it was inhibitedat 41 C

(Fig. 9). SS DNAsynthesisresumed at30 C in

this mutant and at both temperatures in a

tem-perature-insensitive revertant (data not

shown). The same observations were made

us-ing the dnaG mutant host (data not shown).

These experiments serve as controls, showing

thatunder these conditions mutant hosts

defec-tive in late SS DNA synthesis are unable to

resume SS DNA synthesisat 41 C.

RF template DNA accumulation during

X174 infection. Since the dnaB and dnaC

proteins are notrequired for the reinitiation of

nor forthe continuation of SS DNA synthesis

once ithasbegun, the mostlikelyexplanation

for the observed sensitivity of SS synthesis to

early shifts to 41 C in these mutants is the indirect effect of the inhibition of RF template

DNA synthesis. Additional observations

con-cerning 4X174 RF and SS DNAsynthesis in the

dnaC mutant host are consistent with this con-clusion. We uniformly labeled 4X174 DNA in

infected cells throughout infection with

[3H]thymine

andfollowed the accumulation of

label into RF and SS DNA (distinguished by

zonesedimentation). Previous experimentshad

shown that the rate of45X174RF DNA

replica-tion in a dnaC mutant host at 30 C was 15-fold

less thantherate in atemperature-insensitive

revertant (6). Similarly, the data in Fig. 10

show thattheaccumulation of[3H]thymine

la-belinto RF DNA inthednaCmutantLD332 at

30 C wasabout 20-fold slower than in the

tem-perature-insensitive revertant and the parent

hoststrain (compare Fig. 10C to 10Band 10A,

respectively;note thescale change in Fig. 10C).

Therateof SSDNAsynthesiswasalso

propor-tionately slower. In addition, Fig. 10C shows that RF DNAsynthesis continued for atleast 90minafter infectionat 30 Cinthe dnaC mu-tant host. Other experiments showed that RF

DNAcontinuedto be synthesizedatthe initial

rateforatleast2hafterinfection.Incontrast,

RF DNA synthesis occurred rapidly early in

infectioninthenondefectivehosts, and slowed down late in infection. These dataare consist-ent with the conclusion that the RF template

concentration is limiting throughout the

infec-tion in the dnaC mutant host. Shifts to 41 C

limitfurther RF DNA synthesis. Thus, the

ear-lierthe shiftto 41C, the less the concentration

of RFtemplateDNA, the slowerthe rate of SS

DNA synthesis. The same explanation seems

mostlikelyfor the sensitivity of SS DNA

syn-thesis to early shifts to 41 C in the dnaB

mu-tant.

DISCUSSION

Previous experiments have shown that the host cell dnaB and dnaC proteins are not

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SYNTHESIS OF SINGLE-STRANDED

OX174

DNA 433

'0

'°o~~I

I

I

III

0~

4

I - j

20

40

60

20

40

60

FRACTION NUMBER

FIG. 8. Zone sedimentation of pulse-labeled intracellular 4X174 DNA extracted from LD332 (dnaC's). This experimentwassimilartothatdescribedinthelegendtoFig.4,except that the pulse included 200

10Ci

of

[3H]thymidinerather than100MCi,andthechloramphenicol was added (and the temperature shifted up)at

80 minafter infection and removedat120 min.(A) Pulse-labeledat30Cat75 min; (B) pulse-labeledat 41C

at115min;(C)pulse-labeledat41 Cat150min;(D)pulse-labeledat30Cat 190 min.

needed for thecontinuationofSSDNA

synthe-sis once it has begun (3, 6). The experiments

described here show that SSDNAsynthesiscan

reinitiate at 41 C in the nondefective parent

host strain H502 and in the

temperature-sensi-tivednaB anddnaCmutanthost strains. Prior

to the removal of the chloramphenicol from

these infected mutantcells,whichtriggers the

reinitiationof SS DNAsynthesis,SS DNA

syn-thesis is notdetectable. The elevated

tempera-ture (41 C) inactivates the

temperature-sensi-tiveproteins, resultingininhibition of RF

rep-licationinthe mutant cells but not inthe par-ent host cells. Upon removal of the drug, SS

DNA ismadeatnear normal rates.These

crite-ria indicate that the reinitiation of SS DNA

synthesis in our experiments is analogous to

the normal initiation of the SS DNAsynthesis

stage. Assuming this to be the case, we

con-clude that the host cell dnaB and dnaC

pro-teins are not required for the initiation of SS

DNA synthesis.

We observed in our experiments with the

mutant hosts that the maximum rates of SS

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434 DUMAS AND MILLER

IE C'J 6

la0 U.

0

0

E .2 w

rI-TIME

(min)

FIG. 9. Ratesof

OX1

74DNAsynthesisat30and41 Cinhost strainLD301 (dnaEts).At 30 and 60 min

afterinfection, chloramphenicol wasaddedtoportionsofthe cultureat afinalconcentrationof30

pg/ml.

These same portions weresimultaneously shiftedto41 C. At 45minaftertheshiftsthedrugwasremovedby filtrationat 41 C. Portions(2ml)werepulse-labeledwith10 uCiof3H-thymidine. Symbols: *,rate at30C,

nochloramphenicoladded; 0,rate at41 C,shiftedat30min,chloramphenicolremovedat75min;A,rateat 41 C,shiftedat60min,chloramphenicol removedat105 min.

A

14I I I

40_

I I I I I I

I I I I

B

0.05k

I

10.04

I I I

0.03

I I

' 0.02

It

I I

- 0.71

H

,F_

C

I _

I _

I I

I

-I I I

I

40 80 120 40 80 120 40 80 120

TIME(min)

FIG. 10. Relativeamountsof

OX1

74RF DNAand SSDNAsynthesizedat30C in H502, inaspontaneous temperature-insensitiverevertantofLD332,and in LD332. Mitomycin C-treated infected cellswereuniformly

labeled with [3H]thymine(10 MACi/ml)at30Cbeginningatthetimeofinfection. Attheindicatedtimescells

from 20-mlportionswerecollected, washed, lysed,proteasedigested, and subjectedtozonesedimentationin

sucrosedensity gradients. TheamountsofradioactivityintheSS and RF DNA bandsweredetermined and

normalized to the amount in SS DNA in H502 after 120 min. A, H502; B, spontaneous temperature-insensitiverevertantofLD332; C, LD332. Symbols: *,RFDNA; A,SSDNA.

DNA synthesis after reinitiation are greater

the later the simultaneous shift to 41 C and

addition of chloramphenicol. RF replication

continues at 30C throughout the time period

overwhich these temperatureshifts were

exe-cuted. Thusthe later theshift, thegreaterthe

concentration of RF DNA template. The ob-servedincrease inmaximum rates of SS DNA

1.01-F-0.8

z

> 0.6

1 0.4

0.2_

0.0

I I

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those described here (data not shown). Under

these conditions no appreciable SS DNA

syn-thesiswouldhaveoccurredprior to theremoval of thedrug. We found that cells that are kept at

30C canachieve normal rates of SS synthesis uponremovalof the drug, but that RF

replica-tion is required after removal of the drug to

achieve these rates. RF replication is slow at 30C inchloramphenicol. Upon shifting to41 C,

atthetimeofremoval of thechloramphenicol, we observed only slow SS DNA synthesis

lim-itedby theconcentration ofRFtemplateDNA.

To allow accumulation of enough RFtemplate DNA toachieve normal rates of SS DNA syn-thesiswithout furtherRFreplication,weadded thechloramphenicol late enough such that SS DNAsynthesishad begun.

Weconclude from these observations that the dnaB and dnaC proteinsare notdirectly

essen-tial for either the initiation of SSDNA

synthe-sis or itscontinuation. Thereducedratesof SS

DNA synthesis observed at41 C in these mu-tantcells, especially when the temperaturewas

shifted up to 41 Cearly ininfection, are most

likelydue to inhibition of the synthesis of the RFDNAtemplate needed for SS DNA

synthe-sis.

The experiments described here also show that RF DNA replication does not

necessarily

cease atthe time of the initiation of SS DNA synthesis (10). In normal host cellsmostof the

RF DNA is

synthesized early

in

infection,

and

mostof the SS DNA is

synthesized

late.

How-ever, inthednaC mutantcells RF DNA

repli-cation continues atits

early

rateforatleast2h

afterinfection, whereas SSDNA

synthesis

be-gins as early as 30 min after infection. RF

replication and SS synthesis are nottherefore mutually exclusive.

Rather,

in normal

infec-tionsthe saturatinglevel ofRF DNA is

appar-ently

achieved

early

in

infection,

at about the

same time as the onset ofSS DNA

synthesis.

Butthere doesnot seem tobeanecessary

exclu-This work was supported by Public Health Service research grant AI-9882 and research career development award AI-70,632 (to L.B.D.) from the National Institute of Allergyand Infectious Diseases.

LITERATURE CITED

1. Carl, P. L. 1970. Escherichia coli mutants with temper-ature-sensitive synthesis of DNA. Mol. Gen. Genet.

109:107-122.

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

3. Dumas, L. B., and C. A. Miller. 1974. Inhibition of bacteriophage 4X174DNAreplication in dnaB mu-tantsofEscherichia coli C. J. Virol. 14:1369-1379. 4. Hirota, Y., A.Ryter, and F. Jacob. 1968.

Thermosensi-tive mutants of E. coli affected in the processes of DNA synthesis and cellular division. Cold Spring Harbor Symp. Quant. Biol. 33:677-694.

5. Hutchison, C. A., and R. L. Sinsheimer. 1966. The process of infection with bacteriophage 4X174. X. Mutations in a XX lysis gene. J. Mol. Biol. 18:429-447.

6. Kranias, E.G., and L. B. Dumas. 1974. Replication of

bacteriophage4bX174 DNA in a temperature-sensi-tive dnaC mutant ofEscherichia coli C. J. Virol. 13:146-154.

7. Levine, A. J., and R. L.Sinsheimer. 1969. The process of infection withbacteriophage X174. XXV.Studies

withbacteriophage4oX174mutantsblocked in prog-eny replicative form DNA synthesis. J. Mol. Biol. 39:619-639.

8. McFadden, G., and D. T. Denhardt. 1974. Mechanism ofreplicationof4X174 single-strandedDNA. IX. Re-quirement for theEscherichia coli dnaG protein. J. Virol. 14:1070-1075.

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

10. Schubach, W. H., J. D. Whitmer, and C. I. Davern. 1973. Genetic control of DNA initiation in Escher-ichia coli. J. Mol. Biol. 74:205-221.

11. Sinsheimer, R. L., R.Knippers, and T. Komano. 1968.

Stagesinthe replicationofbacteriophage4X174in vivo. ColdSpring Harbor Symp. Quant.Biol. 33:443-447.

12. Wechsler, J. A.,and J. D. Gross.1971.Escherichia coli mutants temperature-sensitive for DNA synthesis.

Mol. Gen. Genet. 113:273-284.

13. Wickner,S., and J. Hurwitz. 1974. Conversion ofqbX174 viral DNA todouble-stranded form bypurified Esch-erichia coli proteins. Proc. Natl. Acad. Sci. U.S.A. 71:4120-4124.

on November 10, 2019 by guest

http://jvi.asm.org/

Figure

FIG. 2.41atatshiftedrate75 41 30 C min Rates of XX1 74 DNA synthesis at 30 and in host strain LD332 (dnaCls)
FIG. 3.portions3041colchloramphenicolratemin C. minm Rates of 4X1 74 DNA synthesis at 30 and 41 C in the parent host strain H502
FIG. 4.portionportionfilteredH502.(D).the Zone sedimentation ofpulse-labeled intracellular 4X174 DNA extracted from parent host strain At 25 min after infection a portion ofthe culture was pulse-labeled at 30 C (A)
FIG. 5.after10Theseremoved75 min, min; Rates ofXX1 74 DNA synthesis at 30 and 41 C in host strain LD311 (dnaB's)
+5

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