Intermediates involved in the replication of double-stranded  phiX174 RF DNA have been identified and partially characterized. Analysis of pulse-labeled RF DNA suggests that the synthesis of progeny RF molecules involves, in part, the addition of nucleotides to linear complementary strands on a circular parental strand as template, so as to produce intermediate DNA strands of greater than viral length. Electron microscopy reveals DNA rings with "tails" and "double rings," which could be the intermediate structures. A model is postulated for the replication process

Knippers, Rolf

Sinsheimer, Robert L.

Whalley, J. Millar

English

Caltech Authors - Main

The process of infection with bacteriophage phiX174, XXX. Replication of double-stranded phiX DNA

THE PROCESS OF INFECTION WITH BACTERIOPHAGE OX174,
XXX. REPLICATION OF DOUBLE-STRANDED 45X DNA *
BY ROLF KNIPPERS,t J. MILLAR WHALLEY, AND ROBERT L. SINSHEIMER
DIVISION OF BIOLOGY, CALIFORNIA INSTITUTE OF TECHNOLOGY, PASADENA
Communicated June 30, 1969
Abstract.-Intermediates involved in the replication of double-stranded 4X174
RF DNA have been identified and partially characterized. Analysis of pulse-
labeled RF DNA suggests that the synthesis of progeny RF molecules involves,
in part, the addition of nucleotides to linear complementary strands on a circular
parental strand as template, so as to produce intermediate DNA strands of
greater than viral length. Electron microscopy reveals DNA rings with "tails"
and "double rings," which could be the intermediate structures. A model is
postulated for the replication process.
Semiconservative replication of the replicative form (RF)1 of bacteriophage
4X174 DNA is only observed for those RF molecules which are associated with
an active "replication site."2' There are one to four replication sites per host
cell,2 dependent on the metabolic state of the cell. In cells infected with mul-
tiplicities of more than four phage per cell, only the parental RF molecules can
participate in the semiconservative production of daughter RF molecules.3
The replicating parental RF molecules are found in the membrane fractions of a
cell extract while the replication products, the daughter RF molecules, are not
bound to the membrane and are most probably free in the cytoplasm of the cell.4
These processes are observed during the eclipse period of a normal infection
cycle and during abortive infection in the presence of 30-40 yg/ml chloramphen-
icol (when only daughter RF molecules, but no progeny single-stranded DNA
molecules, are formed). In this paper we describe some experiments which
give, in a preliminary way, more insight into the process of the semiconservative
reproduction of double-stranded 4X DNA molecules.
Experimental Procedures.-Bacteria and phages: The host strain E. coli H502 (hcr-
Endol-thy-su-) was kindly provided by Dr. Hoffman-Berling. The lysis-defective
mutant, 0Xam3, has been described.' The DNA of the extended host range, tempera-
ture-sensitive mutant of 0Xtsyh, was used as a biological marker. The preparation of
P32-oXam3 has been described.6 The spheroplast assay was that of Guthrie and Sin-
sheimer.7 Mitomycin pretreatment of the host cells to preferentially suppress the synthe-
sis of host cell DNA has been described.8
Media: Minimal medium has been described by Knippers, Razin, Davis, and Sin-
sheimer.9 Tris-EDTA is 0.05 M Tris-HCl-0.005 M EDTA, pH 8.
Infection procedure: E. coli H502 cells at a concentration of 5 X 101 cells/ml were incu-
bated at 370 in the presence of mitomycin. After 30-40 min the cells were resuspended
in fresh medium containing 1 ,ug/ml of thymine and 35 Mg/ml of chloramphenicol. After
5 min at 370, P'2-kXam3 in a multiplicity of 5-6 was added. Five minutes later the cul-
ture was placed in a 200 waterbath. After 20-25 min, H3-thymidine (11 C/mmole,
Schwarz Bioresearch) in a concentration of 0.03 mC/ml was added for various lengths of
time as indicated in the Results section. Replication was terminated by pouring the cul-
ture into a container with an acetone-dry ice mixture at -40°. The cells were then
275
BIOCHEMISTRY: KNIPPERS ET AL.
pelleted in the cold and washed two times with cold Tris-EDTA containing 0.05 M
sodium azide.
Other techniques: The use of sarkosyl and pronase for the extraction of OX RF has
been described.9 Radioactively labeled DNA was precipitated with an equal volume of
cold 10% trichloroacetic acid, collected on glass filter discs, dried, and counted in a scin-
tillation spectrophotometer with a toluene base scintillator. All centrifuge techniques
were those of Knippers and Sinsheimer.'0 Details will be given in the legends to the
appropriate figures. Chromatography on columns of benzoylated, naphthoylated DEAE-
cellulose was performed as previously described.9, 13
Results.-Pulse experiments: A total of 6 X 1010 H502 cells in 100 ml medium
(1 ,ug/ml of thymine, 35 jLg/ml of chloramphenicol) was infected with P32-am3
(multiplicity 5). After 20 minutes at 200, 2 mC H3-thymidine were added for
about 5 seconds. The extracted DNA was first sedimented through a neutral
sucrose gradient (Fig. 1). Parental P32-label appeared in three peaks, corre-
sponding to single-stranded DNA (25S; originating, probably, from adsorbed
phage particles which had not penetrated), to RFI (21S), and to RFII (16.5S).
The H3-pulse label cosedimented predominantly with the P32-RFII, while a
faster-sedimenting shoulder of H3-labeled DNA sedimented with 20-22S. Less
than 10 per cent of the total acid-precipitable H3-radioactivity sedimented with
10S, perhaps analogous to Okazaki's fragments" of replicating E. coli DNA or
T4 bacteriophage DNA.
Fractions 8-16 of the gradient of Figure 1, containing most of the pulse label,
were combined and dialyzed against Tris-EDTA to remove the sucrose. The
DNA in these fractions was then concentrated by isopropanol precipitation in
the presence of denatured calf thymus DNA (0.2 mg) as carrier. The recovered
DNA (about 70%) was denatured (by addition of 0.02 vol 10 N NaOH) and
sedimented through an alkaline sucrose gradient (Fig. 2). About 65 per cent of
the P32-parental label sedimented with the rate of the infectious DNA marker
~ u_
° 2 2 FIG. 1.-Sedimentation analysis of
x x < pulse-labeled, double-stranded
-OX
E E I / \ DNA at neutral pH. Sedimentation
00
C\J
was performed through linear 20-5%
"Is l l Esucrose gradients in 0.3 M NaCl-
Q. EL T Tris-EDTA, in the SW25.1 rotor of
o Q Q /the Spinco centrifuge, for 14 hr at
I~I X zz,25,000rpm at 40. The arrow indi-
cates the direction of sedimentation.
A , ., Z >/ \/tFractionvolume: 1.2 ml.
Fraction Number
276 PROC. N. A. S.
BIOCHEMISTRY: KNIPPERS E7T AL.
FIG. 2.-Sedimentation of pulse- 0 i
labeled OX RF through an alkaline _ l
sucrose gradient. Alkali-denatured c
DNA in a 0.5-ml volume was sedi- 6 6i
mented through a 28-ml gradient (20- E W>
5% sucrose in 0.3 M NaOH-0.01 M 5
-V
EDTA) in the SW25.1 rotor for 27 hr 50a
at 25,000 rpm at 5°. The arrow indi- >
cates the direction of sedimentation. a-
Fraction volume: 0.7 ml. C
lo 20 30 40
Fraction Number
(16S) and was presumably circular DNA (vide infra); approximately 15 per cent
of the P32-counts were on the bottom of the centrifuge tube (probably in fast-
sedimenting, denatured RFI); the remaining P32-radioactivity (15%) sedi-
mented with 14S, the sedimentation coefficient of linear OkX DNA molecules of
unit length.
The H3-pulse label was distributed in a more complex manner. The most
prominent peak of H3-labeled DNA sedimented with 14S, the rate of linear 4X
DNA strands of unit length. Faster-sedimenting pieces of pulse-labeled DNA
appeared in a leading shoulder of this main peak, with sedimentation rate up to
18S, the rate expected for double-length XX DNA strands. DNA pieces smaller
than unit length were also found, sedimenting with S values below 14S. In the
experiment of Figure 2 as well as in other similar experiments, more pulse label
appeared in the fractions of "smaller" DNA pieces than in the "larger" (35%
vs. 15% of the total Ht-acid-precipitable DNA).
All H3-labeled DNA species in the gradient of Figure 2 were degraded by
exonuclease J.12 The only enzyme-resistant DNA found was the P'2-labeled
circular 16S-parental strand. Repeatedly, some 5-20 per cent of the parental
P32-DNA has been found to be in the linear form (14S). Whether such molecules
are artifacts of extraction or whether they represent intermediate states in the
4X replication cycle cannot yet be decided. The variability observed in the
relative amount of linear parental strands in different experiments would favor
the first possibility.
To obtain more information about the nature of the various pulse-labeled DNA
strands, appropriate samples were recovered from the alkaline sucrose gradient
and analyzed by sedimentation to equilibrium in a CsCl gradient at pH 12.5.
The 14S material and the pulse-labeled material in the leading shoulder (S > 14)
usually gave satisfactory distribution profiles at equilibrium; however, no clear
results could be obtained by equilibrium centrifugation of the shorter (S < 14)
fragments. As can be seen from Figure 3, the H3-14S peak contained material
forming bands at the densities of complementary and viral DNA strands in
VOL. 64, 1969 277
BIOCHEMISTRY: KNIPPERS ET AL.
500
E
0L
0
I 250
N
100
E
0.
50
to
-30 40 50 60-97 1-35 40 50 60 65-97
Fraction Number
FIG. 3.-Equilibrium centrifugation of DNA in CsCl at pH 12.5. A 2.7-ml
volume (initial density: 1.75 gm/ml) was centrifuged for 24 hr at 38,000 rpm
and for 48 hr at 30,000 rpm in the SW65 rotor of the Spinco centrifuge at 140.
97 fractions were collected directly on GF/A glass fiber filters, dried, and counted.
(a) Centrifugation of fractions 18-20 of Fig. 2 (S values >14S); (b) Centrifuga-
tion of fraction 22-24 of Fig. 2 (S values around 14S). Density increases from
right to left; V, viral strand; C, complementary strand.
approximately equal amounts, whereas the larger DNA strands (S > 14) were
found predominantly at the density corresponding to complementary strand
material.
Chromatography on benzoylated, naphthoylated DEAE-cellulose (BNC): BNC
columns have been used successfully to separate replicating DNA complexes
from resting molecules.9' 13 Replicating DNA complexes appear to contain
single-stranded regions14' 15 and are more firmly bound to the column. They
can be eluted only with caffeine, whereas the nonreplicating, wholly double-
stranded DNA molecules are eluted with NaCl. The data of Table 1 indicate
that this distinction was also true for the replicating OX RF: The proportion
of the total incorporated acid-precipitable radioactivity eluted only with caf-
feine decreased with increasing length of H3-pulse, suggesting that the immedi-
ately replicating OX RF complexes were present in a partially denatured form.
Figure 4 shows the elution pattern of OX RF, extracted from a P32-am3-
TABLE 1. Chromatography of pulse-labeled OX RF on benzoylated, naphthoylated DEAE-
cellulose.
Pulse time Recovered Per cent in Per cent in
(sec) p.2 (cpm) NaCl eluate caffeine eluate
10 5,210 32 68
40 5,050 34 66
120 4,870 29 71
Recovered
H3 (cpm)
10 1,180 28 72
40 3,705 53 47
120 14,765 69 31
H502 cells (5 X 108/ml) in 30 ml minimal medium (35 jg/ml of chloramphenicol) were infected
with P3-am3 in a multiplicity of 6. After 20 min at 300, Hs-thymidine was added and equal samples
were withdrawn from the culture at the indicated times. The extracted OX DNA was chromato-
graphed as described in the legend to Fig. 4. Final recovery of acid-precipitable radioactivity was
83, 79, and 86% after the 10-, 40-, and 120-sec pulses, respectively.
I
a v c b v c _
IJ <>\booOO>o
278 PROC. N. A. S.
BIOCHEMISTRY: KNIPPERS ET AL.
0
l 100
0.
C)
10 20 25 30 40~~~~~~-50
Fraction Number
FIG. 4.-Chromatography on a column of benzoylated, naphthoylated DEAE-
cellulose. Column dimensions: 2 cm X 3 cm. The column was washed with
50 ml 0.2 M NaCl-Tris-EDTA before the DNA sample was added to the column.
Elution: (i) linear gradient of 0.2 M NaCl to 1 M NaCl in Tris-EDTA; (ii)
linear gradient of 0 to 1.3% caffeine in 1 M NaCl-Tris-EDTA. Flow rate, 0.5
ml/min; fraction volume, 2 ml; recovery of input acid-precipitable radioactivity,
87%.
infected cell culture which had been pulse-labeled with H3-thymidine for 40
seconds at 300. About 50 per cent of the P32-parental DNA and H3-pulse-
labeled DNA eluted in the NaCl gradient, probably representing nonreplicating
parental RF molecules. The other half of the labeled DNA was eluted with
caffeine, in fractions well separated from the single-stranded marker OX DNA.
Our interpretation that the NaCl-eluted DNA represents nonreplicating DNA
and that the caffeine-eluted material contains replicating RF complexes is sup-
ported by the alkaline sucrose gradients shown in Figures 5 and 6.
Centrifugal analysis of the salt-eluted DNA (Fig. 5) showed that most of the
P32-parental label was present in fast-sedimenting denatured RFI (34S) molecules.
Part of the H3-labeled DNA also sedimented with 34S; however, the greater
portion (about 60%) was denatured to circular (16S) and linear (14S) compo-
FIG. 5.-Sedimentation analysis of 10
_ 5
salt-eluted, denatured DNA. The
salt-eluted DNA of the chromatogram
of Fig. 4 was concentrated by CsCl _ 4 0
equilibrium centrifugation. After di- X x
alysis to remove the CsCl, a 0.3-ml E E
sample was denatured (0.03 ml 10 N 3_ 3
NaOH) and sedimented through a 5 I
linear sucrose gradient (20% to 5% X 2 I
sucrose in 0.3M NaOH-0.01 M EDTA, D
pH 12.5) in the SW41 rotor of the
Spinco centrifuge for 6 hr at 40,000
rpm at 5°. The arrow indicates the
direction of sedimentation. Ai
VOL. 64, 1969 279
BIOCHEMISTRY: KNIPPERS ET AL.
300 _ to RF FIG. 6.-Sedimentation analysis of
_ vthe denatured, caffeine-eluted DNA.
The caffeine-eluted DNA of the chro-D1 200 M matogram of Fig. 4 was concentrated
E by a neutral CsCl equilibrium centrif-
100 - tot ugation, dialyzed, and then denaturedSs / with alkali. The denatured DNA was
ll sedimented as described in Fig. 5,
4 100 except that the centrifugation time was9 hr. One half of each fraction was
INl precipitated on a GF/A glass fiber
0 x10 filter and counted. Portions of theX 3 < other half were neutralized and used
_o for the DNA-DNA hybridization pro-
cedure of Waarnar and Cohen,17 em-
2 _ g _ 50 s .> ploying Millipore filters loaded either
CE lkX with denatured RF (4 pg/filter) or withQ phage-extracted viral DNA (SS, 4
t ig/rml). The total H3-radioactivity re-I_ tamied on these filters after three cyclesN^-- .E9 ~ ' of washings with a low ionic strength
0000OOO000000000000 buffer is shown in the inset.
0 20 30 40
Fraction Number
nents from RFII molecules. These early replication products have been de-
scribed previously"6 and are composed of equal amounts of circular viral and
complementary strands and of linear viral and complementary strands, re-
spectively.
The distribution of label observed after alkaline sedimentation of the caffeine-
eluted DNA (Fig. 6) was similar to that shown for the pulse-labeled DNA of
Figure 2. More than 90 per cent of the parental P32-labeled DNA strands was
circular (16S). The H3-pulse label appeared in a prominent 14S-peak and in
faster-sedimenting DNA pieces (S > 14) with a shoulder at 18S (where XX DNA
of twice the unit length is expected to sediment). A shallow peak of H3-labeled
material sedimenting slower than unit length linear DNA (S < 14) was also
present.
The nature of the various pulse-labeled DNA strands was studied by a DNA-
DNA annealing technique.17 The results (inset, Fig. 6) indicated that most of
the slower-sedimenting pieces were composed of sequences present only in the
viral strand of the RF.
Electron microscopy:18 The caffeine-eluted DNA was also examined by electron
microscopy.19 The results of a more systematic study of replicating RF inter-
mediates will be published elsewhere. Two morphological states of replicating
RF have been found in the caffeine eluate, as shown in Figure 7: rings with tails
and "double rings." Tails longer than one unit length of OX DNA were not
detected. From these preliminary data it is not possible to decide which of the
two forms represent the normal morphology of RF intermediates. Hirt's work
on replicating polyoma DNA molecules" would suggest that the "double ring"
structure is the usual form of replicating "small" DNA rings.
Discussion and Summary.-Despite several uncertainties, it appears that a list
of the components of replicating RF molecules should include (1) circular parental
280 PROC. N. A. S.
BIOCHEMISTRY: KNIPPERS ET AL.
(a)
(b)
FIG. 7.-Electron micrograms of replicating AX RF molecules.
DNA strands; (2) linear complementary strands which probably grow by addi-
tion of nucleotides to one of their ends; and (3) DNA pieces of newly synthesized
viral DNA strands which are shorter than one unit length OX DNA.
281VOL. 64, 1969
BIOCHEMISTRY: KNIPPERS ET AL.
r Z Z A membrane
origin
c origin
(a) (b) (c)
FIG. 8.-Model of double-stranded OX RF replication. (a) At
beginning of replication; (b) partially replicated: nascent "tail"
free; (c) partially replicated; nascent "tail" bonded to origin; v,
viral strand; c, complementary strand.
If the enzymatic replication apparatus is located on the host cell membrane,2'
it can be assumed that the circular parental strand rotates through that complex
(Fig. 8). Nucleotides are added to the 3'-OH-terminus of the complementary
strand, thereby producing complementary strand pieces longer than one unit
length. The new viral strand is synthesized in an unknown way on the emerg-
ing old part of the complementary strand. An entangling of the nascent double-
stranded DNA tail should be avoided by a loose association (via a protein?) of the
end with the "origin" of the DNA circle, as suggested by the electron micro-
grams.
* This research was supported in part by grant GM 13554 from the U.S. Public Health
Service.
t Present address: Max-Planck-Institut fuir Med. Forschung, Heidelberg, West Germany.
I RF is the intracellular double-stranded "replicative form" DNA of 4X174. RFI is the
supercoiled DNA duplex; RFII is the "nicked" relaxed form. The RF, containing the DNA
of the infecting virus, is called parental RF. DNA strands, homologous to those found in the
viral particle, are called viral strands; their complementary partners are called complementary
strands.
2 Yarus, M., and R. L. Sinsheimer, J. Virology, 1, 135 (1967).
3 Sinsheimer, R. L., R. Knippers, and T. Komano, in Cold Spring Harbor Symposia on
Quantittive Biology, vol. 33 (1968), p. 443.
4 Knippers, R., and R. L. Sinsheimer, J. Mol. Biol., 34, 17 (1968).
6 Hutchison, C. A., and R. L. Sinsheimer, J. Mol. Biol., 18, 429 (1966).
6 Knippers, R., W. 0. Salivar, J. E. Newbold, and R. L. Sinsheimer, J. Mol. Biol., 39, 641
(1969).
7 Guthrie, G. D., and R. L. Sinsheimer, Biochim. Biophys. Acta, 72, 290 (1963).
8 Lindqvist, B. H., and R. L. Sinsheimer, J. Mol. Biot,, 30, 69 (1967).
9 Knippers, R., A. Razin, R. Davis, and R. L. Sinsheimer, J. Mol. Biol., in press.
I0 Knippers, R., and R. L. Sinsheimer, J. Mol. Biol., 35, 591 (1968).
1" Okazaki, R., T. Okazaki, K. Sakape, K. Sugimoto, and A. Sugiro, these PROCEEDINGS, 59,
598 (1968).
12 Lehman, I. R., and A. L. Nussbaum, J. Biot. Chem., 239, 2628 (1964).
13 Kiger, J. A., and R. L. Sinsheimer, J. Mot. Biol., 40, 467 (1969).
14 Kidson, C., J. Mot. Biol., 17, 1 (1966).
" Smith, M. G., and K. Burton, Biochem. J., 98, 229 (1966).
16 Knippers, R., T. Komano, and R. L. Sinsheimer, these PROCEEDINGS, 59, 577 (1968).
17 Waarnar, S. O., and J. A. Cohen, Biochem. Biophys. Res. Commun., 24, 554 (1966).
18 The assistance generously provided by R. Davis and D. Clayton, California Institute of
Technology, is gratefully acknowledged. The electron micrograms shown here are those of
R. Davis.
"I Kleinschmidt, A. K., and R. K. Zahn, Z. Naturforsch., 14b, 770 (1959).
F 2 Hirt, B., J. Mol. Biol., 40, 141 (1969).
P 21Jacob, F., S. Brenner, and F. Cuzin, in Cold Spring Harbor Symposia on Quantitative
Biology, vol. 28 (1963), p. 329.
282 PRoc. N. A. S.


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The process of infection with bacteriophage phiX174, XXX. Replication of double-stranded phiX DNA

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