In a previous article,(1) it was demonstrated that of the labeled thymidine incorporated into the replicative form (RF) of phiX-DNA during the period of RF replication (3-12 or 15 min after infection), approximately half was transferred to virus during the subsequent period of single-strand DNA synthesis, while half remained in RF molecules. In this paper experiments are presented which permit a definition of the label remaining in the RF and which clarify the role of the RF in the synthesis of the progeny single-stranded DNA

Knippers, Rolf

Komano, Tohru

Sinsheimer, Robert L.

English

Caltech Authors - Main

THE PROCESS OF INFECTION WITH BACTERIOPHAGE OX174,
XXII. SYNTHESIS OF PROGENY SINGLE-STRANDED DNA*
BY TOHRU KOMANO, ROLF KNIPPERS, AND ROBERT L. SINSHEIMER
DIVISION OF BIOLOGY, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA
Communicated December 20, 1967
In a previous article,' it was demonstrated that of the labeled thymidine
incorporated into the replicative form (RF) of OX-DNA during the period of
RF replication (3-12 or 15 min after infection), approximately half was trans-
ferred to virus during the subsequent period of single-strand DNA synthesis,
while half remained in RF molecules. In this paper experiments are presented
which permit a definition of the label remaining in the RF and which clarify the
role of the RF in the synthesis of the progeny single-stranded DNA.
Experimental.-Most of the relevant techniques were described in our preceding
article.'
Exonuclease III digestion: Digestions with exonuclease III2 were performed in 0.05 M
tris buffer, pH 8.0, in the presence of 5 mM MgCl2 and 2 mM mercaptoethanol for 4 hr
at 370C. Approximately 800 units of exonuclease III were added for 50 ,ug DNA in a
total volume of 1.4 ml.
DNA-DNA hybridization: The annealing technique of Denhardt3 was used. Solutions
of labeled DNA (containing about 1 ,ug DNA) were annealed with treated nitrocellulose
filters (HAWP, Millipore) previously loaded with 100 ,ug unlabeled viral OX-DNA,
washed, and counted.
Results.-The nature of the persistent RF label: If RF is labeled with H3-
thymine during the period of RF replication and the label is subsequently chased,
approximately one half of the label remains in the RF.I When cells were
infected with 4Xam3 at 300, labeled with H3-thymine during the period 5-18
minutes after infection, and the label subsequently chased with cold thymine
for 60 minutes, nearly one half the label remained in RF, distributed as 4 RFII: 1
RFI. When this RFII was centrifuged to equilibrium in alkaline CsCl (Fig. 1),
all of the label was found at the buoyant density of the complementary strand.
When this RFII was analyzed by zone sedimentation through a preformed
alkaline CsCl gradient (Fig. 2), approximately 80 per cent of the label sedi-
mented at the rate of the ring form of OX-DNA, while 20 per cent sedimented at
the rate of the linear form.
Evidently, in this RFII, which contained the bulk of the label remaining after
the chase, the label was exclusively in the complementary strand, very largely
in the form of a closed ring.
In a similar experiment, in which cells were labeled with H3-thymine during
the period 0-14 minutes after infection, at 370C, and the label was then chased
with cold thymine for 20 minutes, again about one half the label remained in
RF (6 RFII: 1 RFI). When this RFII was analyzed by zone sedimentation
through a preformed alkaline CsCl gradient, the persistent label was found
(Fig. 3a) partly in circular DNA, partly in whole linear DNA, and partly in
DNA fragments, presumably from RFII molecules with more than one cut in a
strand. When the circular and whole linear DNA components from the gradient
of Figure 3a were separately centrifuged to equilibrium in alkaline CsCl (Figs.
911
BIOCHEMISTRY: KOMANO ET AL.
3b, c), the label was found, in both cases, almost exclusively at the buoyant den-
sity of complementary DNA.
This experiment confirms the result of that previously described, in that the
label persisting in RF is found in the complementary strands, but indicates that
there may be some variability in the extent to which these complementary
strands remain intact, dependent upon experimental conditions.
l ~~~~~~~~~~~~I
V C
a
20 Z FIG. 1-Analysis of RFII isolated
by a zone sedimentation (through a
neutral CsCl gradient) of DNA ex-
z tracted from cells labeled during theCL / A\ \ ~ ° F period of RF replication (5-18 min
nI 100 _ / after infection at 300C) and subse-
f quently "chased" for 60 min. Equi-
. librium distribution in alkaline CsCl/ he,, gradient (OXyh-DNA added as
^^^^^^^ ' ' SJF"~( marker).
25 30 35 40 45 ma77
FRACTION NUMBER
I o i6200
-04
0
4 (1_
LL_ Z 73 ~~~WI
00~~~~~~~~~~~~~~~~~~_a.otoo FIG. 2.-Distribution of H-labelin RFII of Fig. 1 after sedimenta-
to7 W tion through a preformed alkaline
bes~l gradient(oXfyh-DNA added
as marker).
0 10 20 30 40 50
FRACTION NUMBER
Label incorporated into "late" RFII: Lindqvist and Sinsheimer4 showed that
Hi-thymine incorporated during the period of single-strand progeny DNA
synthesis was first detected in RFJJ, before transition to single-stranded DNA.
When such pulse-labeled RFIJ from cells (labeled with H3-thymidine for 1 min at
300C at 45 min after infection) is analyzed by zone sedimentation through a
preformed alkaline CsCl gradient (Fig. 4a), it can be seen that all the label is to
be found in a component sedimenting with the velocity of linear OX-DNA.
When this component is centrifuged to equilibrium in an alkaline CsCl density
gradient (Fig. 4b), all the label is found at the buoyant density of viral clX-
DNA.
Evidently the label incorporated into RFJJ during the period of progeny
single-strand DNA formation is present only in the viral strand, which is open
at one link.
912 PROC. N. A. S.
VOL 59, 1968 BIOCHEMISTRY: KOMANO ET AL. 913l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 1.27 X|0-1790
SO sn \ | L780D0 W b 70>
300 I-
-10
200 80so 160 - 0.4 - 400
N ~ FATO 0.BE 0RCIO.UME260 40 t0l1 300
100 co for 20 m. 0 0 0 3 4 200 ,
FIG. 3.-Analysis of RFII isolated by
zone sedimentation (through a neutral CsCl LW0gradient) of DNA extracted from cells _ 0 _ 1
- 1.790labeled during the period of RF replication -6 7-(0-14 min after infection at 3700) and sub- X
sequently chased for 20 mmn. c}70
(a) Distribution of H3-label after sedi-
mentation through a preformed alkaline -1750 03
CsCl gradient (P32-(pX-DNA added as 200- 0.5-- 500 14
marker). 400
(b) Equilibrium distribution in an alka- 160- 0.3 00
line CsCl density gradient of the "circular" 4012 .
-zoocomponent from Fig. 2a (M. lysodeikticus A
DNA (A20) and P32-tX-DNA added as CA 202 3 0
80 - 0.2-N00cmarkers).
At the end of a short pulse of H3-thymine during this period of single-strand
z~~~~~~~~~~~~~
DNA formation, a small fraction of incorporated label is found as RFI. To
examine the distribution of label in this component, such "late" pulse-labeled
RFI was isolated from a zone sedimentation through neutral CsCl, treated with
sufficient pancreatic deoxyribonuclease to produce an average of one singl0strand
break per molecule, and the product centrifuged to equilibrium in an alkaline
CsCl density gradient. As can be seen in Figured. the label is to be found only
in denatured, unattacked RFI (40%) and in DNA with the buoyant density of
viral OX-DNA (60%). Evidently only the viral strand is labeled in the pulse-
labeled RFI as well as in the RFII.
Digestion with exonuclease III: If the open viral strand of "late" pulse-
labeled RFII has a free 3'OH end, it should be susceptible to digestion by exo-
nuclease III. When such an RFIJ preparation (21,000 cpm/ml or approxi-
mately 5 Ag/ml) was digested with exonuclease III for four hours at 370C,
the acid-precipitable label decreased by 88 per cent. During this incubation the
infectivity5 of the solution increased some sevenfold6 during the first two hours
BIOCHEMISTRY: KOMANO ET AL. PRoCc. N. A. S.
.800
800 b -1790j
|150001|780
80 so 1.770 >-I'
1600 0.5 -1.760
Fn
70 70 1.7500a
~~~~~~~1000 .41.740
C. 160 460
400 It
FT 50
500 0.2-I
200
formation~ ~ ~ ~ ~~~~4-a45m ferifcto)
0.1
30- 30
0 10 20 30 40 50 6020 0 40 5
FRACTION NUMBER FRACTION NUMBER
FIG. 4.-Analysis of RFII (isolated by zone sedimentation through a preformed neutral
CsCl gradient) labeled in a 1-min pukse at 30'C during the period of progeny single-strand DNA
formation (at 45 min after infection).
(a) Distribution of H3-label after zone sedimentation through a preformed alkaline CsCl
gradient (P32-RFII, uniformly labeled, added as a marker).
(b) Equilibrium distribution in alkaline CsCl density gradient of "linear" component from
Fig. 4a (M. lysodeikticus DNA (A260) and P32-RFII, uniformly labeled and randomly nicked,
added as density markers).
s,,
FIG. 5.-Distribution of label in
RFI, isolated by zone sedimentation
M 200 through a preformed neutral CsCl\RF V C gradient of the DNA extracted after
a. > a 1-min pulse ol H3-thyrmine during
-.-. vtheperiod of progeny single-strand
I K00 DNA formation. This RFI wastoto /0 Jbriefly treated with DNase and the
II \productcentrifuged to equilibriumin an alkaline CsCl density gradient
(P32-4X-DNA added as a marker).
1 30 35 40 45 50 90
FRACTION NUMBER
(as a consequence of the release of the more highly infective, single comple-
mentary strands) and then remained constant, indicating that the enzyme
preparation contained no significant endonuclease impurities.
These experiments demonstrate that the open viral strand of the late pulse-
labeled RFII has a free 3'OH end. They also indicate that in the bulk of the
pulse-labeled RFII, the complementary strands must be closed (Fig. 2) or else
the digestion with exonuclease III could not have proceeded essentially to com-
pletion.
Hybridization experiments: Although the alkaline equilibrium density
914
BIOCHEMISTRY: KOMANO ET AL.
gradients of Figures 4b and 5 indicate that most of the label incorporated into
RF during the period of single-stranded DNA formation goes into the viral
DNA strands, a small incorporation into complementary strands could not
be detected by these methods. To test this possibility, pulse-labeled late
RFII (labeled for 1 min at 30'C at 45 min after infection) was denatured and
annealed with a nitrocellulose filter carrying 100 ,tg of unlabeled viral DNA.
Control experiments were performed with labeled viral DNA and with denatured
"artificial" RFII, isolated after deoxyribonuclease digestion of labeled "early"
RFI, of equal specific labeling in both strands. As can be seen in Table 1,
TABJ.E 1. DNA-viral DNA hybridization.
Per cent
Cpm Cpm Per cent complementary
Test DNA added annealed* annealed strands
,X-DNA 320,000 <550 <0.02 0
"Artificial" RFIIt 112,000 2570 2.3T 50
"Late" pulse-labeled RFII 65,000 122-222 0.19-0.34 4.6-8.5
* Background has been subtracted.
t "Artificial" RFII is prepared by light DNase digestion of uniformly labeled "early" RFI.
t In a uniformly labeled RF, 2.3% of the label corresponds to 5.4% of the complementary
strands.
between 0.19 per cent and 0.36 per cent of the label in late RFII anneals to the
filter, as compared to 2.3 per cent of the label of the "artificial" RFII and less
than 0.02 per cent of the viral DNA. From this data and allowing for the
difference in thymine contents of the viral and complementary strands, it may
be calculated that between 4.6 per cent and 8.5 per cent of the strands labeled
during a late pulse are complementary strands.
Discussion.-From the results presented in this and previous papers,1' 7
three phases of DNA replication may be discerned during infection with bacte-
riophage cX174.
In phase I the single-stranded DNA ring is converted to a double-stranded
DNA ring (RF), which may be open or closed (RFII or I). Thisg process is
carried out by pre-existing host-cell enzymes7 and has been duplicated in vitroA8
In phase II, semiconservative replication of the double-stranded DNA ring
involving, frequently, only the RF at a bacterial "site"9 is observed. This
process requires the participation of viral-specified function. 10 In this repli-
cation only the RFII form is active;" the parental DNA strand remains at the
"site," and the nascent RF appears as RFII4 of two types.'
In phase III, the viral strands of the progeny RF are repeatedly displaced
into virus particles with the concomitant synthesis of new viral strands upon
the persistent complementary strands. In the RFII pulse-labeled during this
process the viral strands are open, the complementary strands in greater part
are closed. Evidently the viral strands must be closed at some stage before
completion of the virus particles. During this phase a low level of continuing
synthesis of complementary strands can be detected, possibly a consequence
of a small, continued semiconservative RF replication at the "site."
Sunmmary.-That label which, after incorporation into RF during the period
of RF replication, persists in RF after a period of progeny single-strand DNA
VOL. 59, 1 96ii 9154
BIOCHEMISTRY: KOMANO ET AL.
formation is only in the complementary strands of RF. These strands are,
under some circumstances, largely closed.
Label-incorporated into RFII (or I) during pulses in the "late" period of
single-stranded DNA synthesis is almost exclusively in viral strands, which are
open. These strands in RFII have a free 3'OH end and the complementary
strands of such RFII molecules are for the most part closed.
A low rate of synthesis of complementary strands continues during the period
of progeny single-strand DNA formation.
We are indebted to Dr. M. Goulian for a generous gift of purified exonuclease III and
to Mrs. Louise Wilson for technical assistance.
* This research was supported in part by grant GM 13554 from the U.S. Public Health
Service.
1 Knippers, R., T. Komano, and R. L. Sinsheimer, these PROCEEDINGS, 59, 577 (1968).
2 Richardson, C. C., I. R. Lehman, and A. Kornberg, J. Biol. Chem., 239, 251 (1964).
a Denhardt, D. T., Biochem. Biophys. Res. Commun., 23, 641 (1966).
4Lindqvist, B. H., and R. L. Sinsheimer, J. Mol. Biol., submitted for publication.
6 Guthrie, G. D., and R. L. Sinsheimer, Biochim. Biophys. Acta, 72, 290 (1963).
6Burton, A., and R. L. Sinsheimer, Science, 142, 962 (1963).
7Sinsheimer, R. L., C. A. Hutchison, III, and B. H. Lindqvist, "Bacteriophage OX174:
Viral functions," in The Molecular Biology of Viruses, ed. J. S. Colter and W. Paranchych
(New York: Academic Press, 1967).
8 Goulian, M., A. Kornberg, and R. L. Sinsheimer, these PROCEEDINGS, 58, 2321 (1967).
9 Yarus, M., and R. L. Sinsheimer, J. Virology, 1, 135 (1967).
10 Tessman, E., J. Mol. Biol., 17, 218 (1966).
"Knippers, R., and R. L. Sinsheimer, J. Mol. Biol., submitted for publication.
PROC. N. A. S.916


The process of infection with bacteriophage phiX174, XXII. Synthesis of progeny single-stranded DNA

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The process of infection with bacteriophage phiX174, XXII. Synthesis of progeny single-stranded DNA

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