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 ofsingle-stranded
DNAsynthesis
were measured after theremoval of chloramphenicol thathad been
added
at varioustimesafter infectionto
specifically
stop this stage of 4X174 DNA synthesis. Reinitiation was notdefective ineithermutanthost. Our data
suggested
that the reinitiation of thesingle-stranded
stageof 4X174 DNAsynthesis
intheseexperiments
wasanalo-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 normalinitiationofthe 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 evenduring
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 ofbacteriophage
OX174
on thesingle-stranded (SS)
viral DNA templateinvitro (9, 13).Both the dnaB and dnaC proteinsare
essen-tial for
4X174
DNAsynthesis
invivo(3,6). The synthesis of this phage DNA occurs in three stages (11).Neither proteinisdirectly
involvedinthe continuationof
OX174
SS DNAsynthe-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
SSDNAsynthesis.
We asked ifeither the dnaB or the dnaC
protein of E. coli is directly essential for the
initiation of
4X174
SSDNAsynthesis. Wepres-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 at41C. 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 (200jig)
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. Sarkosylwasaddedto3%, 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 at37 C. The
samples
were then heated for 20 minat 12-56 C.
32P-labeled
4X174
viral DNA marker wasadded. The samples were then layered onto 36-ml
lineargradientsof 5to20%sucrosein50 mMpotas-o l
sium
phosphate-2
mM EDTA-1 MNaCl,
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 wereT 8 ° ° %
shifted from
30to41 C.Symbols: *,
rateat30C;
0,2 * 3% ~ rateat41 C,shiftedat0min;A,rateat41 C,shifted
N I at30
min;
,rate at41 C,shiftedat60 min;V,
rateX
' 5 1 at41 C,shiftedat75 min.3%
bfi
6-faX174
1 | t \am3
lysiss
defective) DNAsynthesis
inU 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 of0 1lXt
ry \
[3H]thymidine
incorporated
into smallportions
40842- AdoftheAd *
mitomycin
C-treatedinfected cell cultureE I
b.
during2-minpulses. The results from aOX174-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 fort2 - /p.r > & and% Ad about 40 min after infection and then
slowly
d
% declined. Most of the DNAsynthesized
at 30Ct1/ X after
approximately
20 to 30 minpostinfection
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 synthesisTIME(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
acomparable experiment
p b e n45 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 30C,
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[image:2.503.272.421.65.334.2] [image:2.503.47.242.306.598.2]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
ofchloramphenicolper 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 additionofchloram-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 minat41C the cultures were filtered. The cellswere
suspended in medium without
chlorampheni-J. VIROL.
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[image:3.503.102.385.58.303.2]SYNTHESIS OF SINGLE-STRANDED
OX174
DNA429
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.VOL. 18, 1976
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[image:4.503.83.407.124.517.2]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
notshown).
Figure4shows thezonesedimentation
anal-ysesof the
OX174
DNAsynthesized
inanother culture at various times during this kind of experiment. Three distinct viral DNA species could be seen intheseanalyses:
SS DNA andthe two
double-stranded
forms RFI and RFII(closed,
circularsupercoils
and open, relaxedcircles, 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
causedtheeventual 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-tionof4X174
SS DNAsynthesisat 41 C.Figure 6A shows that the 25 min after infection inanother.culture
at30 C,immediately
priortoatemperature 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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[image:5.503.112.380.66.297.2]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.
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[image:6.503.112.398.141.533.2]432 DUMAS AND MILLER
24p
9%~~~~~~~~~~~~ 0 016
18
~~~~~~~~~it
I0
1060 120 180
TIME
(min)
FIG. 7. Ratesof
qX1
74DNAsynthesisat30and41 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 oflabel 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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[image:7.503.63.255.53.318.2]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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[image:8.503.105.412.55.473.2]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 minafterinfection, 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
I10.04
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 cellswereuniformlylabeled 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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[image:9.503.144.384.70.291.2] [image:9.503.115.404.363.539.2]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
ininfection,
andmostof the SS DNA is
synthesized
late.How-ever, inthednaC mutantcells RF DNA
repli-cation continues atits
early
rateforatleast2hafterinfection, whereas SSDNA
synthesis
be-gins as early as 30 min after infection. RFreplication and SS synthesis are nottherefore mutually exclusive.
Rather,
in normalinfec-tionsthe saturatinglevel ofRF DNA is
appar-ently
achievedearly
ininfection,
at about thesame 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.