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Initiation and termination sites of adenovirus 12 DNA replication
VIROLOGY
78,
415-424
Initiation
(1977)
and Termination HIROYOSHI
Department
of Tumor
Sites of Adenovirus ARIGA
Virus Research, Shirokane-dni, Accepted
HIROTO
AND
Institute Minato-ku, January
of
12 DNA Replication SHIMOJO
Medical Science, University Tokyo 108, Japan
of
Tokyo,
4-6-1,
23,1977
Initiation and termination sites of adenovirus 12 DNA replication were determined with temperature-sensitive mutants which were defective in the initiation of DNA replication at a restrictive temperature. After a short period of labeling with PHlthymidine, viral DNA molecules labeled at either initiation or termination sites were isolated and cleaved with a restriction endonuclease, either Eco RI or Barn HI. The highest radioactivities were found at both ends of DNA, showing that the initiation and termination sites are located at or near both ends of the genome. The examination of replicating DNA labeled for various times revealed that viral DNA replication initiated at either the right or left end of the genome and proceeded to the opposite end.
sitive (ts) mutants which were defective in initiation of viral DNA replication at the restrictive temperature. The data indicate that Ad 12 DNA replication initiates and terminates at both ends of the molecules, and that DNA replication proceeds from each end to the opposite end of the genome.
INTRODUCTION
Sussenbach and co-workers examined the replication of adenovirus type 5 (Ad 5) DNA and presented the “displacement replication model” (Sussenbach et al., 1972). In this model Ad 5 DNA replication starts at the right end (AT-rich) by displacing the parental heavy (h) strand with continuous polymerization in the 5’ to 3’ direction on the light (1) strand template. After a delay, replication starts on the displaced strand at various internal points, or it may start at the 3’ end of the template molecules in a pattern of continuous growth in the opposite direction to that on the first strand (Ellens et al., 1974; Sussenbath et al., 1974). Recently, it has been reported that the termination sites of Ad 2 DNA replication are both ends of the genome (&hilling et al., 1975; Tolun and Pettersson, 1975; Horwitz, 1976). Furthermore, Horwitz (1976) reported that the pattern of termination label shows an asymmetry in radioactivity on the separated strands of each restriction endonuclease fragment and suggested that there is an initiation point placed at or near each end of the molecule. In this study we examined directly the initiation and termination sites of Ad 12 DNA replication, using temperature-sen-
MATERIALS
AND
METHODS
Cells and viruses. Secondary cultures of human embryo kidney (HEK) cells and monolayer cultures of KB cells were used. The wild type (WT) and two ts mutants of Ad 12, defective in initiation of viral DNA replication at a nonpermissive temperature (40”) (ts A 275, ts B 221) (Shiroki and Shimojo, 1974) were used. The conditions of infection and the nature of the mutants have been previously described (Shiroki et al., 1972; Shiroki and Shimojo, 1974). The infected cells were placed in 1 ml of medium in a small plugged bottle and were incubated in a water bath at either 34 f 0.1” (permissive temperature) or 40 +- 0.1” (nonpermissive temperature) to assure rapid cooling or warming. Radioactive labeling of cells. The labeling of the initiation sites of Ad 12 DNA (initiation label) was carried out as follows. HEK cells were infected with ts A 415
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6622
416
ARIGA
AND
275 or ts B 221 at an input multiplicity of 20 PFU/cell at 34”. At 30 hr postinfection (p.i.), the cells were shifted up to 40”. After 2 hr at 40”, the cells were shifted down to 34” and simultaneously labeled with 13Hlthymidine (23.0 Ci/mmol) at 0.2 or 1 mCi/ml for the times indicated under Results. The newly synthesized viral DNA was extracted by the Hirt procedure (Hirt, 1967). The labeling of the termination sites of Ad 12 DNA (termination label) was carried out as follows. HEK cells were infected with ts A 275 or ts B 221 at 34” as described above. At 30 hr p.i., the cells were shifted up to 40”. After 20 min, cells were labeled with 13Hlthymidine at 0.2 or 1 mCi/ml for 5, 10, or 30 min. The newly synthesized viral DNA was extracted by the Hirt procedure. Purification of viral DNA. The initiation-labeled viral DNA in the Hirt supernatant was diluted with 2 vol of 10 mM Tris-HCI (pH 7.4) and 5 mM EDTA, extracted once with phenol saturated with 10 n&f Tris-HCl (pH 8.1), twice with chloroform-isoamylalcohol (24:1, v/v), and precipitated with 2 vol of ethanol at -20 overnight. The precipitate was collected by centrifugation at 10,000 g for 10 min, dissolved in 1 ml of 0.3 M NaCl, 10 mM TrisHCI (pH 8.1), and 10 miV EDTA, and loaded onto 2-ml columns of benzoyl-naphthoyl-DEAE (BND)-cellulose (Serva). The double-stranded (ds) DNA was eluted with 1 M NaCI, 10 miV Tris-HCl (pH 8.1), and 10 mM EDTA, and the DNA containing single-stranded (ss) regions was eluted with 1 M NaCl, 2% caffeine, 10 mM TrisHCl (pH 8.1), and 10 miV EDTA (van der Eb, 1973). DNA containing ss regions was pooled, precipitated with 2 vol of ethanol and redissolved in the endonuclease digestion buffer described below. The termination-labeled viral DNA in the Hirt supernatant was centrifuged in 5 to 20% neutral sucrose gradients containing 0.5 M NaCI, 50 mM Tris-HCl (pH 7.5), and 10 miV EDTA in an SW 27 rotor for 16 hr at 22,000 rpm at 4”. The 31 S fractions were pooled, precipitated with 2 vol of ethanol, and dissolved in 1 ml of 0.3 M NaCI, 10 mit4 Tris-HCl (pH 8.1), and 10 mM EDTA. Double-stranded DNA was
SHIMOJO
eluted with BND-cellulose scribed.
column
as de-
Hydroxylapatite column chromatography. The ratio of ds to ss DNA in pulse-
labeled DNA was examined by hydroxylapatite (HA) column chromatography. Aliquots (50 to 100 ~1) of the Hirt supernatant containing newly synthesized viral DNA were diluted in 4 ml of 0.12 M Naphosphate (pH 6.8), and treated with a Kubota MS II-250 sonic oscillator at full power for 10 min at 4”. The size of the fragments was 600 to 800 nucleotide pairs (approximately 7.7 S) as estimated by analytical ultracentrifugation. The solutions received sodium dodecyl sulfate (SDS, flnal 0.4%). “Batch elution” from HA (BioRad HTP, DNA grade) was carried out as described by Fujinaga et al. (1975). Alkaline sucrose gradients. The newly synthesized viral DNA in the Hirt supernatant was loaded onto 5 to 20% alkaline sucrose gradients in 0.3 N NaOH, 0.5 M NaCl, and 10 mEA EDTA, and centrifuged in an SW 27 rotor at 22,000 rpm for 16 hr at 4”. Fractions were collected from the bottom, and trichloroacetic acid (TCA)-insoluble counts were examined. Restriction endonucleases. The enzyme from Escherichia coli RY 13 (Eco RI) was kindly provided by Drs. Y. Sugino and T. Kurokawa, Central Research Division, Takeda Chemical Industries Ltd., Osaka, Japan. Endonuclease digestion was carried out for 4 hr at 37” in digestion buffer (90 mit4 Tris-HCI, pH 7.9, 10 n&f MgCl,). Bacillus amyloliquefaciens H (Barn HI) was kindly provided by Drs. T. Ando and T. Shibata, Institute of Physical and Chemical Research, Wako-Shi, Saitama, Japan, and purified by the method of Wilson and Young (1975). Endonuclease digestion was carried out for 4 hr at 37” in digestion buffer (6 n-&f Tris-HCl, pH 7.4, 6 mM MgC12, 6 n&f 2-mercaptoethanol). The reaction was stopped by the addition of EDTA (final 20 mM). Then glycerol (10%) and bromophenol blue (0.1%) were added. The digested DNA fragments were separated by electrophoresis on 2.5% polyacrylamide column gel (0.6 x 12 cm) or 1.4% agarose (Seakern, Marine Colloids, Inc.) column gel (0.6 x 25 cm) in TrisEDTA-acetate buffer (40 miV Tris-ace-
ADENOVIRUS
DNA
INITIATION
tate, pH 7.4, 20 mM sodium acetate, 2 mM EDTA) for 16 hr at 4 V/cm. The former gels were sliced at 1 mm. Each slice was melted in 0.5 ml of H,O, at 80” overnight and then counted in a Triton-toluene-based scintillator. The latter gels were stained with ethidium bromide (1 pg/ml) and the DNA bands were visualized with uv light. Then, the portion of the gel with the visualized band was cut out and counted as described above.
of Labeled DNA in Cells with Ad 12 ts Mutants
All experiments described here were carried out with Ad 12 ts mutants. Since
5
IO Fraction
TERMINATION
417
the results were the same between cells infected with ts A 275 and ts B 221 mutants, only the results with the ts A mutant are described. At 30 hr p.i., the cells were pulse labeled with [3H]thymidine for 5 min at a permissive temperature (34”). Then, the cells were shifted up to a nonpermissive temperature (40”) and chased after addition of cold thymidine for 2 hr. Viral DNA was separated from cellular DNA by the Hirt procedure. The Hirt supernatants containing viral DNA were centrifuged on alkaline sucrose gradients (Fig. 1). Figure 1A (5min pulse) showed two peaks, completed molecules and replicating molecules. Figure 1B (5-min pulse plus 2-hr chase) shows only completed molecules. The replicating molecules of adeno-
RESULTS
Examination Infected
AND
15
20
25
30
Number
FIG. 1. Growth of Ad 12 DNA at a nonpermissive temperature. HEK cells infected with Ad 12 fs A 275 were pulse labeled with 13Hlthymidine for 5 min at 30 hr p.i. at 34”. Then, the cells were washed two times, shifted up to 40”, and chased with cold thymidine (0.1 m&f) for 2 hr. Viral DNA from the Hirt supernatant was centrifuged on alkaline sucrose gradients as described under Materials and Methods. The ratio of ds to ss DNA in an aliquot of the Hirt supernatants was examined by HA chromatography as described under Materials and Methods and is shown in the inserts of this figure. (A) B-min pulse at 34”, (B) 5-min pulse at 34” and chase for 2 hr at 40”. 0, 3H-Labeled viral DNA in the Hirt supernatant; A, 3*P-Labeled Ad 12 DNA from purified virions (a marker).
418
ARIGA
AND SHIMOJO
virus DNA were known to contain ss regions of relatively large extents (Pettersson, 1973; Sussenbach et al., 1973; van der Eb, 1973). The proportion of ss to ds DNA was therefore examined by HA chromatography and is shown in the insert in Fig. 1. The relatively large proportion (23.7%) of ss DNA labeled during a short pulse reduced to the smaller portion (4.2%) after the chase at 40” for 2 hr. When the marker 32P-labeled Ad 12 DNA was examined with HA, 96% of native DNA eluted as ds, while 97% of heat-denatured DNA eluted as ss. These results show that viral DNA replication is completed during the 2-hr chase at 40”. No new initiation occurs at 40” as reported previously (Shiroki and Shimojo, 1974). After 2 hr at 40”, the cells were shifted down again to 34”, pulse-labeled with 13H]thymidine for 5, 10, 20, 40, 60, or 90 min, and the viral DNA was analyzed in alkaline sucrose gradients (Fig. 2). This experiment was carried out to label newly initiated DNA at 34”. The sizes of viral DNA became larger with time. The time for completion of one round of DNA replication under this condition was 60 to 90 min. The ratio of ds to ss DNA was examined (Fig. 3). After a 5-min pulse, more than half (68%) of the labeled DNA was ss. The ratio decreased rapidly. After 40 min, the ratio of ss DNA became nearly constant (20%); this may be replicating Ad 12 DNA.
4
FIG. 2. Initiation and growth of Ad 12 DNA at a permissive temperature. HEK cells infected with Ad 12 ts A 275 at 34” were shifted up to 40” at 30 hr p.i. After 2 hr at 40”, the cells were shifted down to 34” and simultaneously labeled with PHlthymidine for various times. Viral DNAs in the Hirt supernatant were analyzed in alkaline sucrose gradients as described under Materials and Methods. 0, 5 min; A, 10 min; 0, 20 min; A, 40 min; x , 60 min; 0, 90 min. The arrow shows the position of the marker DNA (34 53).
100 t
c
Cleavage of Pulse-Labeled DNA with Restriction Endonucleases Eco RI and Barn HI
The shifted-down and pulse-labeled rH]DNA at 34” was extracted and mixed with uniformly labeled [32PlDNA from Ad 12 virions. The DNAs were digested with the restriction endonuclease Eco RI and the fragments were separated by electrophoresis on 2.5% polyacrylamide gel (Fig. 4). The physical map of Ad 12 DNA by Eco RI has been determined (Mulder et al., 1974; Ortin et al., 1976), and is shown on the upper right part in Fig. 4A, where the Eco RI fragments are designated in capital letters (A to E). After a 5-min pulse, only the fragments A and C were labeled. A small peak of label between the fragments D and E may be a growing but not com-
Pulse
Ttme
(min.)
FIG. 3. The proportion of ss DNA in pulse-labeled viral DNA. Aliquots of pulse-labeled viral DNA in Fig. 2 were reduced to fragments by sonication. ds and ss DNA in each aliquot were examined by HA chromatography as described under Materials and Methods. The proportion of ss DNA in total labeled DNA is expressed in percentages.
ADENOVIRUS
DNA
ABC II
I
INITIATION
AND
419
TERMINATION
D I
Slice
Number
FIG. 4. Polyacrylamide gel electrophoresis of Eco RI digest of pulse-labeled DNA. The pulse-labeled viral DNA in the Hirt supematant was prepared under the conditions described in Fig. 2 (5- and lo-min pulses), extracted and mixed with uniformly 32P-labeled Ad 12 DNA from virions. The mixture was digested with Eco RI. Gels were sliced after electrophoresis. Each slice was melted and the radioactivity in each slice was counted. The physical map of Ad 12 DNA examined by Eco RI is shown in the upper right part of the figure. (A) 5-min pulse; (B) lo-min pulse. 0, 3H-Labeled viral DNA; 0, 32P-labeled Ad 12 DNA.
pleted form of the D fragment (Fig. 4A). After a lo-min pulse, all the fragments were labeled, but the ratio of 3H to 32P in the fragment B was smaller than in other fragments. Since both the A and C fragments are mapped at both ends of Ad 12 DNA, this shows that replication of Ad 12 DNA initiates at or near both ends of the genome. DNA labeled by a 5-min pulse was analysed by BND-cellulose column chromatography (Fig. 5). In this experiment 56% of total counts was eluted as DNA containing ss regions. The peak of DNA containing ss regions (fraction 10) was digested with Eco RI and analysed by gel electrophoresis. The fragments A and C were also labeled in this experiment (Fig. 6). The ratio of 3H/32P was higher in the C fragment than in the A fragment, possibly due to the larger size (37% of the genome) of the A fragment than the C fragment
(16%). In order to confirm the above conclusion, the pulse-labeled DNA was digested with another restriction endonuclease, Barn HI (Fig. 7). The physical map of Ad 12 DNA with Barn HI has been determined (Ortin et al., 1976). The highest specific activity (3H/32P) was found at the both ends, in the A and E fragments. The ratio of 3H/32P was higher in the E fragment (right end of the genome) than in the A fragment. This result confums that the replication of Ad 12 DNA initiates at or near both ends of the genome. Dirktion
of DNA
Replication
The change in the proportion of the 3Hlabeled replicating DNA to uniformly labeled DNA was determined at various time after the shift down. Pulse-labeled DNA was mixed with cold Ad 12 DNA and digested with Eco RI. The fragments were separated through a 1.4% agarose gel,
420
ARIGA
AND
stained with ethidium bromide, and cut out. The radioactivity in each fragment was counted. Then, the ratio of the radioactivity in a fragment to the total radioactivity was calculated, and corrected by dividing by the ratio of the molecular weight of each fragment to that of Ad 12 DNA. Based on the assumption that thymine was equally distributed through Ad 12 DNA, the ratio must be 1, when each fragment is equally labeled (Fig. 8). The ratio
SHIMOJO
in the C fragment located on the left end of Ad 12 DNA was highest at 5 min and decreased rapidly with time. The ratio was lowest at 40 min and then approached 1. The ratio in the A fragment located on the right end of Ad 12 DNA was high at 5 min and then approached 1. The ratio in the B fragment located in the middle of the genome was 0 at 5 min, increased to a level higher than 1 at 40 min, and then decreased to 1 at 60 min. The ratio in the D fragment located next to fragment C was 0 at 5 min and rapidly increased to above 1 at 10 min. It decreased again to less than 1 8
~-LL-
-LA.
5 Fraction
IO Number
FIG. 5. BND-Cellulose chromatography of viral DNA labeled at the initiation sites. Viral DNA from cells shifted down and labeled for 5 min as in Fig. 2 was extracted from the Hirt supernatant, loaded onto BND-cellulose, and eluted as described under Materials and Methods. The radioactivity in a 0.2ml aliquot of each fraction (2 ml) was counted. The arrow shows the point of elution buffer change.
I
FIG. 7. Order of labeling in selected regions of ss DNA examined by Barn HI. Viral DNA labeled at the initiation sites as in Fig. 2 (5- and lo-min pulses) was extracted and chromatographed on BND-cellulose column as in Fig. 3. DNA containing ss regions was pooled, mixed with uniformly 32P-labeled Ad 12 DNA and digested with Barn HI. The ratio of 3H/3*P in each DNA fragment was calculated. The ratio is normalized by taking the ratio in the F fragment as 1. The Barn HI map of Ad 12 DNA was determined by Ortin et al. (1976). 0, 5-min; A, lo-min label.
E
0
IO
20
30
40
50 She
60 Number
70
80
90
100
II0
FIG. 6. Polyacrylamide gel electrophoresis of Eco RI digest of viral DNA labeled at the initiation sites and purified by BND-cellulose column chromatography. Viral DNA labeled at the initiation sites, as in Fig. 2, was chromatographed on BND-cellulose column as in Fig. 5. The fraction 10 in Fig. 5 was precipitated with ethanol, dissolved, mixed with uniformly 32P-labeled Ad 12 DNA, digested with Eco RI, and analyzed by polyacrylamide gel electrophoresis as in Fig. 4. 0, 3H-Labeled viral DNA; 0, 32P-labeled Ad 12 DNA.
ADENOVIRUS
DNA INITIATION
3.0 2.8
! ( I’/’ ‘\‘\ \ --_ &, I./ ‘\\ ’ 0 , ---‘Tr--_ / ‘\ 0 1 ’
0.8 F
‘xx,,,
0.6 L 0.4 t
\ z’
”
421
AND TERMINATION
ing replicating and completed viral DNA molecules was centrifuged on neutral sucrose gradients (Fig. 9). The completed molecules (31 S) were pooled and loaded onto the BND-cellulose column (Fig. 10). The ds DNA portion (fraction 2) was mixed with 32P-labeled Ad 12 DNA and digested with either Eco BI or Barn HI. The fragments were separated through 1.4% agarose gel, stained with ethidium bromide, cut out, and counted. The ratio of pulselabeled 13HlDNA to uniformly labeled
P
0.2 0.0
IO
20 30 40 50 60 70 80 90 Pulse
Time
(rn1n.j
FIG. 8. The ratio of radioactivity in each fragment to the radioactivity of the whole DNA molecule. HEK cells were infected with Ad 12 ts A at 34 for 30 hr, shifted up for 2 hr, shifted down and simultaneously labeled for 5,10,20,40,60, or 90 min as in Fig. 2. Pulse-labeled viral DNA was extracted and digested with Eco RI. The fragments were separated through agarose gel and the radioactivity in each fragment was counted. The ratio of radioactivity in a fragment to that of the whole DNA molecules was calculated. The value was divided by the ratio of the molecular weight of the fragment to that of whole DNA. 0, A fragment; A, B fragment; 0, C fragment; n , D fragment.
at 40 min, and then approached 1. This complicated pattern of each fragment suggests that Ad 12 DNA replication initiates at or near each end of the genome and proceeds bidirectionally to the opposite end. This suggestion will be explained schematically in Fig. 12 and under Discussion. Termination tion
Fraction
Number
FIG. 9. Neutral sucrose gradients of the viral DNA labeled at the termination sites. HEK cells (lo” cells) were labeled with 13H]thymidine at the termination sites for 5, 10, and 30 min at 20 min after shiR up to 40”. Viral DNA from the Hirt supernatant was analysed in neutral sucrose gradients as described under Materials and Methods. The radioactivity in an aliquot (0.1 ml) from each fraction was counted. The fraction indicated by the upper bar were pooled. 0,5-min; A, lo-min; l ,30-rein label.
Label ofAd 12 DNA Replica-
The above results suggest that the termination sites of Ad 12 DNA replication are located on both ends. The determination of termination sites was carried out as follows. HEK cells infected with ts A 275 at 34” were shifted up to 40” at 30 hr p.i. Only elongation and no new initiation was observed at 40” (Shiroki and Shimojo, 1974). At 20 min after shift-up, the cells were pulse labeled with [3Hlthymidine for 5, 10, or 30 min. The Hirt supernatant contain-
6 Fracthon
12 Number
FIG. 10. BND-cellulose chromatography of the viral DNA labeled at the termination sites. The fractions indicated by the bar in Fig. 9 were pooled, precipitated with ethanol, dissolved, and chromatographed on a BND-cellulose column as in Fig. 5. The radioactivity of an aliquot (0.2 ml) of each fraction was counted. The arrow shows elution buffer change. 0, 5-min; a, lo-min; 0, 30-min label.
422
ARIGA ~ A. Eco
RI
AND SHIMOJO
~
for 5 min at 30 hr p.i., when replication of Ad 12 DNA was maximum, and treated with the same procedure. The specific activity in each fragment of ds DNA was calculated. The results were the same, showing the high label at both ends of the genome (data not shown). Similar results were reported about Ad 2 DNA (Tolun and Pettersson, 1975; Schilling et al., 1975; Horwitz, 1976; Bourgaux et al., 1976). DISCUSSION
C
1 B. Barn
D
0
A
EF
HI
G
D.H.1
F
C
B
E
FIG. 11. Order of labeling in selected regions of ds DNA. The ds DNA region (fraction 2 in Fig. 101 was precipitated with ethanol, dissolved, mixed with uniformly 32P-labeled Ad 12 DNA, and digested with either Eco RI or Barn HI. Ratio of pulse-labeled [3H]DNA to uniformly labeled [3ZPlDNA were calculated. These results were normalized to give a ratio of 1 for the D fragment (Eco RI) and the F fragment (Barn HI). A digested with Eco RI; B digested with Barn HI. 0, 5-min; A, lo-min; 0, 30-min label.
[32P]DNA was calculated (Fig. 11). The highest specific activity was found in the C and A fragments of 5min labeled DNA (Fig. 11A). The pattern of lo-min labeled DNA was similar to that of 5-min, but the ratio became lower than that of the 5-min label (Fig. 11A). The ratio of 30-min labeled DNA became nearly 1. Similar results were obtained with Bam HI fragments (Fig. 11B). This result suggests that termini of Ad 12 DNA replication are located at or near both ends. KB cells infected with Ad 12 WT were pulse-labeled
Sussenbach et al. (1972) presented the “displacement replication model,” based on the electron-microscopic observation of replicating molecules of Ad 5 DNA. In this model, the replication of Ad 5 DNA starts at the right end of the genome on the 1 strand, displacing the h strand in the form of a single-stranded branch. Recently, it has been reported that the termination sites of Ad 2 DNA are located on both ends of the genome (Tolun and Pettersson, 1975; Schilling et al., 1975; Bourgaux et al., 1976; Horwitz, 19761, based on the analysis of short-pulsed DNA with restriction endonucleases. The original displacement replication model was a little modified by this finding (Levine et al., 1976). Furthermore, Horwitz (1976) reported that the initiation sites are also located on both ends of the genome, based on experiments on shortpulsed Ad 2 DNA with the combination of cleavage with restriction endonucleases and strand separation. However, this conclusion is derived indirectly from analysis of termination-labeled Ad 2 DNA. We could successfully synchronize the initiation and termination of Ad 12 DNA replication using cells infected with Ad 12 ts mutants defective in initiation of viral DNA replication at a restrictive temperature (40”) (Shiroki and Shimojo, 1974). When Ad 12 ts A-infected cells are incubated at a permissive temperature (34”), viral DNA replication initiates and proceeds actively at 30 hr p.i. When the cells are shifted up to 40” and labeled with [3H]thymidine, the termination sites are labeled preferentially without labeling the initiation sites (termination label), since only elongation and termination proceed without new initiation at 40” (Shiroki and Shimojo, 1974). When the shifted-up cells
ADENOVIRUS CD
DNA
INITIATION
A
B
--5 min ---
C and A
high
B
low
c and
D
40 min
high
r:= 60-90
low
___-_----
----
completed
min .3SplCh~O"O"S
FIG. 12. The schematic model of Ad 12 DNA replication. Parental strands were drawn as solid lines and new daughter DNA as dashed lines. The upper lines represent h strands and the lower lines 1 strands. The uppermost letters show the locations of the Eco RI cleaved fragments.
are maintained at 40”, viral DNA synthesis is completed within 2 hr (Fig. 2). When these cells are again shifted down to 34” and simultaneously labeled with [3Hlthymidine for a short time, only the initiation sites are labeled without labeling the termination sites (initiation label). The analysis of these labeled DNA with either Eco RI or Barn HI restriction endonuclease revealed that the initiation as well as termination sites are located at or near both ends of the genome. A modified displacement replication model of adenovirus DNA is presented in Fig. 12. When initiation-labeled DNA (5 min in Fig. 12) is examined, both ends of the genome are highly labeled. If we assume two initiation sites in a single DNA molecule, a DNA molecule with two single-stranded branches at both ends must be visualized by electron microscopy. Electron microscopy of Ad 5 replicating DNA molecules shows only molecules with a single-stranded branch and molecules without any branch (Sussenbach et al., 1972). The above assumption is, therefore, improbable. Half of the replicating mole-
AND
423
TERMINATION
cules may initiate at or near the right end of the genome (right-initiated) as described by Sussenbach et al. (19741, while the remaining half may initiate at or near the left end of the genome (left-initiated). The DNA replication may proceed to the opposite end. At 40 min of labeling in Fig. 12, DNA replication may proceed over the middle of the genome in both right- and left-initiated molecules, while the initiation sites may be labeled only on either the right or the left end of each molecule. This assumption may explain the label higher than 1 in the B fragment located in the middle of the genome, and that lower than 1 in the C or D fragment located on the left end of the genome at 40 min (Fig. 8). This tendency is not so marked in the A fragment of the right end, possibly due to the large size of this fragment (approximately 40% of the genome). At 90 min or longer of labeling, the ratio of the label in each fragment becomes near 1 (Fig. B), possibly due to the completion and asynchrony of viral DNA replication (Fig. 12). Horwitz (1976) observed an asymmetry of the label in the separated strands of each fragment of Ad 2 DNA and suggested the replication of DNA in the 5’ to 3’ direction. The analysis of the separated strands of Ad 12 DNA fragments in our system is now in progress to confirm this idea. Note added. Atter submittal of this manuscript, the initiation sites of Ad 5 DNA (Sussenbach et al., 1976) and of Ad 2 DNA (Weingartner et al., 1976) have been reported. The conclusions in the both reports are in agreement with the present report. ACKNOWLEDGMENTS We are grateful to Dr. K. Shiroki for Ad 12 ts mutants, to Drs. K. Fujinaga and K. Sekikawa for technical instructions, to Drs. Y. Sugino and T. Kurokawa for Eco RI, to Drs. T. Ando and T. Shibata for Barn HI, and to Dr. W. Doerfler, Institute of Genetics, University of Cologne, Cologne, Germany for personal communication of unpublished data. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan and from the Princess Takamatsu Fund for Cancer Research. REFERENCES BOURGAUX, MOISY,
P., DELBECCHI, D. (1976). Initiation
L., and BOURGAUX-RAof adenovirus type
2
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AND SHIMOJO
DNA replication. Virology 72, 89-98. D. J., SUSSENBACH, J. D., and JANZ, H. S. (1974). Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA. Virology 61, 427-442. FUJINAGA, K., SEKIKAWA, K., and YAMAZAKI, H. (1975). Method for determination of nucleotide sequence homology between viral genomes by DNA reassociation kinetics. J. Virol. 15, 466-470. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. ELLENS,
Biol. 26, 365-369. HORWITZ, M. S. (1976).
Bidirectional replication of adenovirus type 2 DNA. J. Virol. 18, 307-315. LEVINE, A. J., VAN DER VLIET, P. C., and SUSSENBACH, J. S. (1976). The replication of papovavirus and adenovirus DNA. Curr. Top. Microbial. Immunol. 73, 67-124. MULDER, C., SHARP, P. A., DELIUS, H., and PETTERSSON, U. (1974). Specific fragmentation of DNA of adenovirus serotype 3, 5, 7, and 12, and adenosimian virus 40 hybrid virus Ad 2+ ND, by restriction endonuclease R Eco RI. J. Virol. 14, 68-77. ORTIN, J., SCHEILWMANN, K-H., GREENBERG, R., WESTPHAL, M., and DOERFLER, W. (1976). Transcription of the genome of adenovirus type 12. III. Maps of stable RNA from productively infected human cells and abortively infected and transformed hamster cells. J. Virol. 20, 355-372. PETTERS~ON, U. (1973). Some unusual properties of replicating adenovirus type 2 DNA. J. Mol. Biol. 81, 521-527. SCHILLING, R., WEING~RTNER,
B., and WINNACKER, E-L. (1975). Adenovirus type 2 DNA replication. II. Termini of DNA replication. J. Virol. 16, 767774. SHIROKI, K., IRISAWA, J., and SHIMOJO, H. (1972). Isolation and a preliminary characterization of
temperature-sensitive mutants of adenovirus 12. 49, l-11. K. and SHIMOJO, H. (1974). Analysis of adenovirus 12 temperature-sensitive mutants defective in viral DNA replication. Virology 61,474485. SUSSENBACH, J. S., VAN DER VLIET, P. C., ELLENS, D. J., and JANSZ, H. S. (1972). Linear intermediates in the replication of adenovirus DNA. Nature
Virology SHIROKI,
New Biol. SUSSENBACH,
239, 47-49. J. S., ELLENS,
D. J., and JANSZ, H. S. (1973). Studies on the mechanism of replication of adenovirus DNA. II. The nature of singlestranded DNA in replicative intermediates. J. Virol. 12, 1131-1138. SUSSENBACH, J. S., ELLENS, D. J., VAN DER VLIET, P. C., KUIJK, M. G., STEENBERGH, P. H., VLAK, J. M., ROZIJN, T. H., and JANSZ, H. S. (1974). The mechanism of replication of adenovirus type 5 DNA. Cold Spring Harbor Symp. Quant. Biol. 39, 539-545. SUSSENBACH,
J. S., TOLUN, A., and PEITERSSON, U. (1976). Nature of the single-stranded DNA in replicating adenovirus type 5 DNA. J. Virol. 20, 532534. TOLUN, A., and PETTERSSON, U. (1975). Termination sites of adenovirus type 2 DNA replication. J. Virol. 16, 759-766. VAN DER EB, A. J.
(1973). Intermediates in type 5 DNA replication. Virology 51, 11-23. WEINGHRTNER, B., WINNACKER, E-L., TOLUN, A., and PETTERSSON, U. (1976). Two complementary strand-specific termination sites for adenovirus DNA replication. Cell 9, 159-268. WILSON, G. A., and YOUNG, F. E. (1975). Isolation of a sequence-specific endonuclease (Barn I) from Bacillus amyloliquefaciens H. J. Mol. Biol. 97, 123-125. adenovirus
78,
415-424
Initiation
(1977)
and Termination HIROYOSHI
Department
of Tumor
Sites of Adenovirus ARIGA
Virus Research, Shirokane-dni, Accepted
HIROTO
AND
Institute Minato-ku, January
of
12 DNA Replication SHIMOJO
Medical Science, University Tokyo 108, Japan
of
Tokyo,
4-6-1,
23,1977
Initiation and termination sites of adenovirus 12 DNA replication were determined with temperature-sensitive mutants which were defective in the initiation of DNA replication at a restrictive temperature. After a short period of labeling with PHlthymidine, viral DNA molecules labeled at either initiation or termination sites were isolated and cleaved with a restriction endonuclease, either Eco RI or Barn HI. The highest radioactivities were found at both ends of DNA, showing that the initiation and termination sites are located at or near both ends of the genome. The examination of replicating DNA labeled for various times revealed that viral DNA replication initiated at either the right or left end of the genome and proceeded to the opposite end.
sitive (ts) mutants which were defective in initiation of viral DNA replication at the restrictive temperature. The data indicate that Ad 12 DNA replication initiates and terminates at both ends of the molecules, and that DNA replication proceeds from each end to the opposite end of the genome.
INTRODUCTION
Sussenbach and co-workers examined the replication of adenovirus type 5 (Ad 5) DNA and presented the “displacement replication model” (Sussenbach et al., 1972). In this model Ad 5 DNA replication starts at the right end (AT-rich) by displacing the parental heavy (h) strand with continuous polymerization in the 5’ to 3’ direction on the light (1) strand template. After a delay, replication starts on the displaced strand at various internal points, or it may start at the 3’ end of the template molecules in a pattern of continuous growth in the opposite direction to that on the first strand (Ellens et al., 1974; Sussenbath et al., 1974). Recently, it has been reported that the termination sites of Ad 2 DNA replication are both ends of the genome (&hilling et al., 1975; Tolun and Pettersson, 1975; Horwitz, 1976). Furthermore, Horwitz (1976) reported that the pattern of termination label shows an asymmetry in radioactivity on the separated strands of each restriction endonuclease fragment and suggested that there is an initiation point placed at or near each end of the molecule. In this study we examined directly the initiation and termination sites of Ad 12 DNA replication, using temperature-sen-
MATERIALS
AND
METHODS
Cells and viruses. Secondary cultures of human embryo kidney (HEK) cells and monolayer cultures of KB cells were used. The wild type (WT) and two ts mutants of Ad 12, defective in initiation of viral DNA replication at a nonpermissive temperature (40”) (ts A 275, ts B 221) (Shiroki and Shimojo, 1974) were used. The conditions of infection and the nature of the mutants have been previously described (Shiroki et al., 1972; Shiroki and Shimojo, 1974). The infected cells were placed in 1 ml of medium in a small plugged bottle and were incubated in a water bath at either 34 f 0.1” (permissive temperature) or 40 +- 0.1” (nonpermissive temperature) to assure rapid cooling or warming. Radioactive labeling of cells. The labeling of the initiation sites of Ad 12 DNA (initiation label) was carried out as follows. HEK cells were infected with ts A 415
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6622
416
ARIGA
AND
275 or ts B 221 at an input multiplicity of 20 PFU/cell at 34”. At 30 hr postinfection (p.i.), the cells were shifted up to 40”. After 2 hr at 40”, the cells were shifted down to 34” and simultaneously labeled with 13Hlthymidine (23.0 Ci/mmol) at 0.2 or 1 mCi/ml for the times indicated under Results. The newly synthesized viral DNA was extracted by the Hirt procedure (Hirt, 1967). The labeling of the termination sites of Ad 12 DNA (termination label) was carried out as follows. HEK cells were infected with ts A 275 or ts B 221 at 34” as described above. At 30 hr p.i., the cells were shifted up to 40”. After 20 min, cells were labeled with 13Hlthymidine at 0.2 or 1 mCi/ml for 5, 10, or 30 min. The newly synthesized viral DNA was extracted by the Hirt procedure. Purification of viral DNA. The initiation-labeled viral DNA in the Hirt supernatant was diluted with 2 vol of 10 mM Tris-HCI (pH 7.4) and 5 mM EDTA, extracted once with phenol saturated with 10 n&f Tris-HCl (pH 8.1), twice with chloroform-isoamylalcohol (24:1, v/v), and precipitated with 2 vol of ethanol at -20 overnight. The precipitate was collected by centrifugation at 10,000 g for 10 min, dissolved in 1 ml of 0.3 M NaCl, 10 mM TrisHCI (pH 8.1), and 10 miV EDTA, and loaded onto 2-ml columns of benzoyl-naphthoyl-DEAE (BND)-cellulose (Serva). The double-stranded (ds) DNA was eluted with 1 M NaCI, 10 miV Tris-HCl (pH 8.1), and 10 mM EDTA, and the DNA containing single-stranded (ss) regions was eluted with 1 M NaCl, 2% caffeine, 10 mM TrisHCl (pH 8.1), and 10 miV EDTA (van der Eb, 1973). DNA containing ss regions was pooled, precipitated with 2 vol of ethanol and redissolved in the endonuclease digestion buffer described below. The termination-labeled viral DNA in the Hirt supernatant was centrifuged in 5 to 20% neutral sucrose gradients containing 0.5 M NaCI, 50 mM Tris-HCl (pH 7.5), and 10 miV EDTA in an SW 27 rotor for 16 hr at 22,000 rpm at 4”. The 31 S fractions were pooled, precipitated with 2 vol of ethanol, and dissolved in 1 ml of 0.3 M NaCI, 10 mit4 Tris-HCl (pH 8.1), and 10 mM EDTA. Double-stranded DNA was
SHIMOJO
eluted with BND-cellulose scribed.
column
as de-
Hydroxylapatite column chromatography. The ratio of ds to ss DNA in pulse-
labeled DNA was examined by hydroxylapatite (HA) column chromatography. Aliquots (50 to 100 ~1) of the Hirt supernatant containing newly synthesized viral DNA were diluted in 4 ml of 0.12 M Naphosphate (pH 6.8), and treated with a Kubota MS II-250 sonic oscillator at full power for 10 min at 4”. The size of the fragments was 600 to 800 nucleotide pairs (approximately 7.7 S) as estimated by analytical ultracentrifugation. The solutions received sodium dodecyl sulfate (SDS, flnal 0.4%). “Batch elution” from HA (BioRad HTP, DNA grade) was carried out as described by Fujinaga et al. (1975). Alkaline sucrose gradients. The newly synthesized viral DNA in the Hirt supernatant was loaded onto 5 to 20% alkaline sucrose gradients in 0.3 N NaOH, 0.5 M NaCl, and 10 mEA EDTA, and centrifuged in an SW 27 rotor at 22,000 rpm for 16 hr at 4”. Fractions were collected from the bottom, and trichloroacetic acid (TCA)-insoluble counts were examined. Restriction endonucleases. The enzyme from Escherichia coli RY 13 (Eco RI) was kindly provided by Drs. Y. Sugino and T. Kurokawa, Central Research Division, Takeda Chemical Industries Ltd., Osaka, Japan. Endonuclease digestion was carried out for 4 hr at 37” in digestion buffer (90 mit4 Tris-HCI, pH 7.9, 10 n&f MgCl,). Bacillus amyloliquefaciens H (Barn HI) was kindly provided by Drs. T. Ando and T. Shibata, Institute of Physical and Chemical Research, Wako-Shi, Saitama, Japan, and purified by the method of Wilson and Young (1975). Endonuclease digestion was carried out for 4 hr at 37” in digestion buffer (6 n-&f Tris-HCl, pH 7.4, 6 mM MgC12, 6 n&f 2-mercaptoethanol). The reaction was stopped by the addition of EDTA (final 20 mM). Then glycerol (10%) and bromophenol blue (0.1%) were added. The digested DNA fragments were separated by electrophoresis on 2.5% polyacrylamide column gel (0.6 x 12 cm) or 1.4% agarose (Seakern, Marine Colloids, Inc.) column gel (0.6 x 25 cm) in TrisEDTA-acetate buffer (40 miV Tris-ace-
ADENOVIRUS
DNA
INITIATION
tate, pH 7.4, 20 mM sodium acetate, 2 mM EDTA) for 16 hr at 4 V/cm. The former gels were sliced at 1 mm. Each slice was melted in 0.5 ml of H,O, at 80” overnight and then counted in a Triton-toluene-based scintillator. The latter gels were stained with ethidium bromide (1 pg/ml) and the DNA bands were visualized with uv light. Then, the portion of the gel with the visualized band was cut out and counted as described above.
of Labeled DNA in Cells with Ad 12 ts Mutants
All experiments described here were carried out with Ad 12 ts mutants. Since
5
IO Fraction
TERMINATION
417
the results were the same between cells infected with ts A 275 and ts B 221 mutants, only the results with the ts A mutant are described. At 30 hr p.i., the cells were pulse labeled with [3H]thymidine for 5 min at a permissive temperature (34”). Then, the cells were shifted up to a nonpermissive temperature (40”) and chased after addition of cold thymidine for 2 hr. Viral DNA was separated from cellular DNA by the Hirt procedure. The Hirt supernatants containing viral DNA were centrifuged on alkaline sucrose gradients (Fig. 1). Figure 1A (5min pulse) showed two peaks, completed molecules and replicating molecules. Figure 1B (5-min pulse plus 2-hr chase) shows only completed molecules. The replicating molecules of adeno-
RESULTS
Examination Infected
AND
15
20
25
30
Number
FIG. 1. Growth of Ad 12 DNA at a nonpermissive temperature. HEK cells infected with Ad 12 fs A 275 were pulse labeled with 13Hlthymidine for 5 min at 30 hr p.i. at 34”. Then, the cells were washed two times, shifted up to 40”, and chased with cold thymidine (0.1 m&f) for 2 hr. Viral DNA from the Hirt supernatant was centrifuged on alkaline sucrose gradients as described under Materials and Methods. The ratio of ds to ss DNA in an aliquot of the Hirt supernatants was examined by HA chromatography as described under Materials and Methods and is shown in the inserts of this figure. (A) B-min pulse at 34”, (B) 5-min pulse at 34” and chase for 2 hr at 40”. 0, 3H-Labeled viral DNA in the Hirt supernatant; A, 3*P-Labeled Ad 12 DNA from purified virions (a marker).
418
ARIGA
AND SHIMOJO
virus DNA were known to contain ss regions of relatively large extents (Pettersson, 1973; Sussenbach et al., 1973; van der Eb, 1973). The proportion of ss to ds DNA was therefore examined by HA chromatography and is shown in the insert in Fig. 1. The relatively large proportion (23.7%) of ss DNA labeled during a short pulse reduced to the smaller portion (4.2%) after the chase at 40” for 2 hr. When the marker 32P-labeled Ad 12 DNA was examined with HA, 96% of native DNA eluted as ds, while 97% of heat-denatured DNA eluted as ss. These results show that viral DNA replication is completed during the 2-hr chase at 40”. No new initiation occurs at 40” as reported previously (Shiroki and Shimojo, 1974). After 2 hr at 40”, the cells were shifted down again to 34”, pulse-labeled with 13H]thymidine for 5, 10, 20, 40, 60, or 90 min, and the viral DNA was analyzed in alkaline sucrose gradients (Fig. 2). This experiment was carried out to label newly initiated DNA at 34”. The sizes of viral DNA became larger with time. The time for completion of one round of DNA replication under this condition was 60 to 90 min. The ratio of ds to ss DNA was examined (Fig. 3). After a 5-min pulse, more than half (68%) of the labeled DNA was ss. The ratio decreased rapidly. After 40 min, the ratio of ss DNA became nearly constant (20%); this may be replicating Ad 12 DNA.
4
FIG. 2. Initiation and growth of Ad 12 DNA at a permissive temperature. HEK cells infected with Ad 12 ts A 275 at 34” were shifted up to 40” at 30 hr p.i. After 2 hr at 40”, the cells were shifted down to 34” and simultaneously labeled with PHlthymidine for various times. Viral DNAs in the Hirt supernatant were analyzed in alkaline sucrose gradients as described under Materials and Methods. 0, 5 min; A, 10 min; 0, 20 min; A, 40 min; x , 60 min; 0, 90 min. The arrow shows the position of the marker DNA (34 53).
100 t
c
Cleavage of Pulse-Labeled DNA with Restriction Endonucleases Eco RI and Barn HI
The shifted-down and pulse-labeled rH]DNA at 34” was extracted and mixed with uniformly labeled [32PlDNA from Ad 12 virions. The DNAs were digested with the restriction endonuclease Eco RI and the fragments were separated by electrophoresis on 2.5% polyacrylamide gel (Fig. 4). The physical map of Ad 12 DNA by Eco RI has been determined (Mulder et al., 1974; Ortin et al., 1976), and is shown on the upper right part in Fig. 4A, where the Eco RI fragments are designated in capital letters (A to E). After a 5-min pulse, only the fragments A and C were labeled. A small peak of label between the fragments D and E may be a growing but not com-
Pulse
Ttme
(min.)
FIG. 3. The proportion of ss DNA in pulse-labeled viral DNA. Aliquots of pulse-labeled viral DNA in Fig. 2 were reduced to fragments by sonication. ds and ss DNA in each aliquot were examined by HA chromatography as described under Materials and Methods. The proportion of ss DNA in total labeled DNA is expressed in percentages.
ADENOVIRUS
DNA
ABC II
I
INITIATION
AND
419
TERMINATION
D I
Slice
Number
FIG. 4. Polyacrylamide gel electrophoresis of Eco RI digest of pulse-labeled DNA. The pulse-labeled viral DNA in the Hirt supematant was prepared under the conditions described in Fig. 2 (5- and lo-min pulses), extracted and mixed with uniformly 32P-labeled Ad 12 DNA from virions. The mixture was digested with Eco RI. Gels were sliced after electrophoresis. Each slice was melted and the radioactivity in each slice was counted. The physical map of Ad 12 DNA examined by Eco RI is shown in the upper right part of the figure. (A) 5-min pulse; (B) lo-min pulse. 0, 3H-Labeled viral DNA; 0, 32P-labeled Ad 12 DNA.
pleted form of the D fragment (Fig. 4A). After a lo-min pulse, all the fragments were labeled, but the ratio of 3H to 32P in the fragment B was smaller than in other fragments. Since both the A and C fragments are mapped at both ends of Ad 12 DNA, this shows that replication of Ad 12 DNA initiates at or near both ends of the genome. DNA labeled by a 5-min pulse was analysed by BND-cellulose column chromatography (Fig. 5). In this experiment 56% of total counts was eluted as DNA containing ss regions. The peak of DNA containing ss regions (fraction 10) was digested with Eco RI and analysed by gel electrophoresis. The fragments A and C were also labeled in this experiment (Fig. 6). The ratio of 3H/32P was higher in the C fragment than in the A fragment, possibly due to the larger size (37% of the genome) of the A fragment than the C fragment
(16%). In order to confirm the above conclusion, the pulse-labeled DNA was digested with another restriction endonuclease, Barn HI (Fig. 7). The physical map of Ad 12 DNA with Barn HI has been determined (Ortin et al., 1976). The highest specific activity (3H/32P) was found at the both ends, in the A and E fragments. The ratio of 3H/32P was higher in the E fragment (right end of the genome) than in the A fragment. This result confums that the replication of Ad 12 DNA initiates at or near both ends of the genome. Dirktion
of DNA
Replication
The change in the proportion of the 3Hlabeled replicating DNA to uniformly labeled DNA was determined at various time after the shift down. Pulse-labeled DNA was mixed with cold Ad 12 DNA and digested with Eco RI. The fragments were separated through a 1.4% agarose gel,
420
ARIGA
AND
stained with ethidium bromide, and cut out. The radioactivity in each fragment was counted. Then, the ratio of the radioactivity in a fragment to the total radioactivity was calculated, and corrected by dividing by the ratio of the molecular weight of each fragment to that of Ad 12 DNA. Based on the assumption that thymine was equally distributed through Ad 12 DNA, the ratio must be 1, when each fragment is equally labeled (Fig. 8). The ratio
SHIMOJO
in the C fragment located on the left end of Ad 12 DNA was highest at 5 min and decreased rapidly with time. The ratio was lowest at 40 min and then approached 1. The ratio in the A fragment located on the right end of Ad 12 DNA was high at 5 min and then approached 1. The ratio in the B fragment located in the middle of the genome was 0 at 5 min, increased to a level higher than 1 at 40 min, and then decreased to 1 at 60 min. The ratio in the D fragment located next to fragment C was 0 at 5 min and rapidly increased to above 1 at 10 min. It decreased again to less than 1 8
~-LL-
-LA.
5 Fraction
IO Number
FIG. 5. BND-Cellulose chromatography of viral DNA labeled at the initiation sites. Viral DNA from cells shifted down and labeled for 5 min as in Fig. 2 was extracted from the Hirt supernatant, loaded onto BND-cellulose, and eluted as described under Materials and Methods. The radioactivity in a 0.2ml aliquot of each fraction (2 ml) was counted. The arrow shows the point of elution buffer change.
I
FIG. 7. Order of labeling in selected regions of ss DNA examined by Barn HI. Viral DNA labeled at the initiation sites as in Fig. 2 (5- and lo-min pulses) was extracted and chromatographed on BND-cellulose column as in Fig. 3. DNA containing ss regions was pooled, mixed with uniformly 32P-labeled Ad 12 DNA and digested with Barn HI. The ratio of 3H/3*P in each DNA fragment was calculated. The ratio is normalized by taking the ratio in the F fragment as 1. The Barn HI map of Ad 12 DNA was determined by Ortin et al. (1976). 0, 5-min; A, lo-min label.
E
0
IO
20
30
40
50 She
60 Number
70
80
90
100
II0
FIG. 6. Polyacrylamide gel electrophoresis of Eco RI digest of viral DNA labeled at the initiation sites and purified by BND-cellulose column chromatography. Viral DNA labeled at the initiation sites, as in Fig. 2, was chromatographed on BND-cellulose column as in Fig. 5. The fraction 10 in Fig. 5 was precipitated with ethanol, dissolved, mixed with uniformly 32P-labeled Ad 12 DNA, digested with Eco RI, and analyzed by polyacrylamide gel electrophoresis as in Fig. 4. 0, 3H-Labeled viral DNA; 0, 32P-labeled Ad 12 DNA.
ADENOVIRUS
DNA INITIATION
3.0 2.8
! ( I’/’ ‘\‘\ \ --_ &, I./ ‘\\ ’ 0 , ---‘Tr--_ / ‘\ 0 1 ’
0.8 F
‘xx,,,
0.6 L 0.4 t
\ z’
”
421
AND TERMINATION
ing replicating and completed viral DNA molecules was centrifuged on neutral sucrose gradients (Fig. 9). The completed molecules (31 S) were pooled and loaded onto the BND-cellulose column (Fig. 10). The ds DNA portion (fraction 2) was mixed with 32P-labeled Ad 12 DNA and digested with either Eco BI or Barn HI. The fragments were separated through 1.4% agarose gel, stained with ethidium bromide, cut out, and counted. The ratio of pulselabeled 13HlDNA to uniformly labeled
P
0.2 0.0
IO
20 30 40 50 60 70 80 90 Pulse
Time
(rn1n.j
FIG. 8. The ratio of radioactivity in each fragment to the radioactivity of the whole DNA molecule. HEK cells were infected with Ad 12 ts A at 34 for 30 hr, shifted up for 2 hr, shifted down and simultaneously labeled for 5,10,20,40,60, or 90 min as in Fig. 2. Pulse-labeled viral DNA was extracted and digested with Eco RI. The fragments were separated through agarose gel and the radioactivity in each fragment was counted. The ratio of radioactivity in a fragment to that of the whole DNA molecules was calculated. The value was divided by the ratio of the molecular weight of the fragment to that of whole DNA. 0, A fragment; A, B fragment; 0, C fragment; n , D fragment.
at 40 min, and then approached 1. This complicated pattern of each fragment suggests that Ad 12 DNA replication initiates at or near each end of the genome and proceeds bidirectionally to the opposite end. This suggestion will be explained schematically in Fig. 12 and under Discussion. Termination tion
Fraction
Number
FIG. 9. Neutral sucrose gradients of the viral DNA labeled at the termination sites. HEK cells (lo” cells) were labeled with 13H]thymidine at the termination sites for 5, 10, and 30 min at 20 min after shiR up to 40”. Viral DNA from the Hirt supernatant was analysed in neutral sucrose gradients as described under Materials and Methods. The radioactivity in an aliquot (0.1 ml) from each fraction was counted. The fraction indicated by the upper bar were pooled. 0,5-min; A, lo-min; l ,30-rein label.
Label ofAd 12 DNA Replica-
The above results suggest that the termination sites of Ad 12 DNA replication are located on both ends. The determination of termination sites was carried out as follows. HEK cells infected with ts A 275 at 34” were shifted up to 40” at 30 hr p.i. Only elongation and no new initiation was observed at 40” (Shiroki and Shimojo, 1974). At 20 min after shift-up, the cells were pulse labeled with [3Hlthymidine for 5, 10, or 30 min. The Hirt supernatant contain-
6 Fracthon
12 Number
FIG. 10. BND-cellulose chromatography of the viral DNA labeled at the termination sites. The fractions indicated by the bar in Fig. 9 were pooled, precipitated with ethanol, dissolved, and chromatographed on a BND-cellulose column as in Fig. 5. The radioactivity of an aliquot (0.2 ml) of each fraction was counted. The arrow shows elution buffer change. 0, 5-min; a, lo-min; 0, 30-min label.
422
ARIGA ~ A. Eco
RI
AND SHIMOJO
~
for 5 min at 30 hr p.i., when replication of Ad 12 DNA was maximum, and treated with the same procedure. The specific activity in each fragment of ds DNA was calculated. The results were the same, showing the high label at both ends of the genome (data not shown). Similar results were reported about Ad 2 DNA (Tolun and Pettersson, 1975; Schilling et al., 1975; Horwitz, 1976; Bourgaux et al., 1976). DISCUSSION
C
1 B. Barn
D
0
A
EF
HI
G
D.H.1
F
C
B
E
FIG. 11. Order of labeling in selected regions of ds DNA. The ds DNA region (fraction 2 in Fig. 101 was precipitated with ethanol, dissolved, mixed with uniformly 32P-labeled Ad 12 DNA, and digested with either Eco RI or Barn HI. Ratio of pulse-labeled [3H]DNA to uniformly labeled [3ZPlDNA were calculated. These results were normalized to give a ratio of 1 for the D fragment (Eco RI) and the F fragment (Barn HI). A digested with Eco RI; B digested with Barn HI. 0, 5-min; A, lo-min; 0, 30-min label.
[32P]DNA was calculated (Fig. 11). The highest specific activity was found in the C and A fragments of 5min labeled DNA (Fig. 11A). The pattern of lo-min labeled DNA was similar to that of 5-min, but the ratio became lower than that of the 5-min label (Fig. 11A). The ratio of 30-min labeled DNA became nearly 1. Similar results were obtained with Bam HI fragments (Fig. 11B). This result suggests that termini of Ad 12 DNA replication are located at or near both ends. KB cells infected with Ad 12 WT were pulse-labeled
Sussenbach et al. (1972) presented the “displacement replication model,” based on the electron-microscopic observation of replicating molecules of Ad 5 DNA. In this model, the replication of Ad 5 DNA starts at the right end of the genome on the 1 strand, displacing the h strand in the form of a single-stranded branch. Recently, it has been reported that the termination sites of Ad 2 DNA are located on both ends of the genome (Tolun and Pettersson, 1975; Schilling et al., 1975; Bourgaux et al., 1976; Horwitz, 19761, based on the analysis of short-pulsed DNA with restriction endonucleases. The original displacement replication model was a little modified by this finding (Levine et al., 1976). Furthermore, Horwitz (1976) reported that the initiation sites are also located on both ends of the genome, based on experiments on shortpulsed Ad 2 DNA with the combination of cleavage with restriction endonucleases and strand separation. However, this conclusion is derived indirectly from analysis of termination-labeled Ad 2 DNA. We could successfully synchronize the initiation and termination of Ad 12 DNA replication using cells infected with Ad 12 ts mutants defective in initiation of viral DNA replication at a restrictive temperature (40”) (Shiroki and Shimojo, 1974). When Ad 12 ts A-infected cells are incubated at a permissive temperature (34”), viral DNA replication initiates and proceeds actively at 30 hr p.i. When the cells are shifted up to 40” and labeled with [3H]thymidine, the termination sites are labeled preferentially without labeling the initiation sites (termination label), since only elongation and termination proceed without new initiation at 40” (Shiroki and Shimojo, 1974). When the shifted-up cells
ADENOVIRUS CD
DNA
INITIATION
A
B
--5 min ---
C and A
high
B
low
c and
D
40 min
high
r:= 60-90
low
___-_----
----
completed
min .3SplCh~O"O"S
FIG. 12. The schematic model of Ad 12 DNA replication. Parental strands were drawn as solid lines and new daughter DNA as dashed lines. The upper lines represent h strands and the lower lines 1 strands. The uppermost letters show the locations of the Eco RI cleaved fragments.
are maintained at 40”, viral DNA synthesis is completed within 2 hr (Fig. 2). When these cells are again shifted down to 34” and simultaneously labeled with [3Hlthymidine for a short time, only the initiation sites are labeled without labeling the termination sites (initiation label). The analysis of these labeled DNA with either Eco RI or Barn HI restriction endonuclease revealed that the initiation as well as termination sites are located at or near both ends of the genome. A modified displacement replication model of adenovirus DNA is presented in Fig. 12. When initiation-labeled DNA (5 min in Fig. 12) is examined, both ends of the genome are highly labeled. If we assume two initiation sites in a single DNA molecule, a DNA molecule with two single-stranded branches at both ends must be visualized by electron microscopy. Electron microscopy of Ad 5 replicating DNA molecules shows only molecules with a single-stranded branch and molecules without any branch (Sussenbach et al., 1972). The above assumption is, therefore, improbable. Half of the replicating mole-
AND
423
TERMINATION
cules may initiate at or near the right end of the genome (right-initiated) as described by Sussenbach et al. (19741, while the remaining half may initiate at or near the left end of the genome (left-initiated). The DNA replication may proceed to the opposite end. At 40 min of labeling in Fig. 12, DNA replication may proceed over the middle of the genome in both right- and left-initiated molecules, while the initiation sites may be labeled only on either the right or the left end of each molecule. This assumption may explain the label higher than 1 in the B fragment located in the middle of the genome, and that lower than 1 in the C or D fragment located on the left end of the genome at 40 min (Fig. 8). This tendency is not so marked in the A fragment of the right end, possibly due to the large size of this fragment (approximately 40% of the genome). At 90 min or longer of labeling, the ratio of the label in each fragment becomes near 1 (Fig. B), possibly due to the completion and asynchrony of viral DNA replication (Fig. 12). Horwitz (1976) observed an asymmetry of the label in the separated strands of each fragment of Ad 2 DNA and suggested the replication of DNA in the 5’ to 3’ direction. The analysis of the separated strands of Ad 12 DNA fragments in our system is now in progress to confirm this idea. Note added. Atter submittal of this manuscript, the initiation sites of Ad 5 DNA (Sussenbach et al., 1976) and of Ad 2 DNA (Weingartner et al., 1976) have been reported. The conclusions in the both reports are in agreement with the present report. ACKNOWLEDGMENTS We are grateful to Dr. K. Shiroki for Ad 12 ts mutants, to Drs. K. Fujinaga and K. Sekikawa for technical instructions, to Drs. Y. Sugino and T. Kurokawa for Eco RI, to Drs. T. Ando and T. Shibata for Barn HI, and to Dr. W. Doerfler, Institute of Genetics, University of Cologne, Cologne, Germany for personal communication of unpublished data. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan and from the Princess Takamatsu Fund for Cancer Research. REFERENCES BOURGAUX, MOISY,
P., DELBECCHI, D. (1976). Initiation
L., and BOURGAUX-RAof adenovirus type
2
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AND SHIMOJO
DNA replication. Virology 72, 89-98. D. J., SUSSENBACH, J. D., and JANZ, H. S. (1974). Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA. Virology 61, 427-442. FUJINAGA, K., SEKIKAWA, K., and YAMAZAKI, H. (1975). Method for determination of nucleotide sequence homology between viral genomes by DNA reassociation kinetics. J. Virol. 15, 466-470. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. ELLENS,
Biol. 26, 365-369. HORWITZ, M. S. (1976).
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