Vol. 16, No. 4 Printed in U.S.A.
JOURNAL OF VIROLOGY, Oct. 1975, p. 838-843 Copyright 0 1975 American Society for Microbiology
Degradation of the Viral Strand of OX174 Parental Replicative-Form DNA in a Rep- Host KATHERINE L. BOWMAN AND DAN S. RAY* Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024 Received for publication 6 May 1975
A progressive degradation of the parental viral strand label is observed upon infection of a Rep- mutant of Escherichia coli by 32P-labeled OX174. Very little parental label remains in the RF (replicative form) by 47 min after infection. Concomitant with this degradation, replicative intermediates are formed which sediment at 21s, the rate of RF I (supercoiled-closed circular DNA), in a neutral sucrose gradient but which denature and sediment in alkaline gradients as single strands of unit size and larger. These denaturable 21s replicative intermediates have been shown previously to be RF molecules containing an elongated viral strand. Addition of chloramphenicol at 7 min after infection at 30 gg/ml, a concentration sufficient to block RF
SS (single strand) synthesis but not RF
-
RF synthesis in a wild-type host cell, reduced the amount of viral strand elongation but did not prevent viral strand degradation. The addition of chloramphenicol at 150,ug/ml at 7 min after infection totally prevents both the degradation of the parental label and the formation of the replicative intermediates with elongated tails. We infer that degradation of the viral strand requires the gene A-mediated nicking of the viral strand but not the concomitant elongation of the viral strand.
The replicative cycle of 4X174 DNA can be divided into three stages: (i) conversion of the parental single-stranded circular DNA to the double-stranded circular replicative form (SS - RF), the initial copy of which is termed the parental replicative form; (ii) RF replication (RF -- RF) in which numerous copies of the RF are made; and (iii) asymmetric synthesis of progeny single-stranded DNA (RF - SS) (5, 8,9). Only a single viral gene product, the gene A protein, is involved in RF - RF synthesis (10). Replication of the parental RF depends on a specific endonucleolytic cleavage of the viral strand of the RF by the OX174 gene A protein (6). In the absence of a functional gene A protein, the parental RF accumulates as RF I and does not replicate further (3). The first stage of replication, SS - RF occurs normally in a Rep- host and is followed by covalent closure of the complementary strand. In the presence of a wild-type gene A, this parental RF molecule is nicked in the viral strand (3). Further replication is blocked in a Rep- host. However, upon prolonged incubation of infected Rep- cells, a small amount of viral strand synthesis occurs by extension of the nicked viral strand (3, 4). This synthesis beyond the block imposed by the Rep - mutation leads
to the formation of intermediate structures sedimenting in neutral gradients at 21s, the same rate as the supercoiled RF I. Yet, upon alkaline sedimentation, these structures yield circular complementary strands and viral strands of greater than unit length. By electron microscopy, these intermediates appear to have a sigma structure, a relaxed duplex ring with a single-stranded tail (4). In this paper, the formation of these 21s denaturable replicative intermediates is discussed with an emphasis on the fate of the viral strand in the presence of a wild-type gene A.
MATERIALS AND METHODS Phage and bacterial strains. The sources and properties of the strains used here have been described previously (2, 3). The Rep- mutant used was Escherichia coli rep, (1). Media and solutions. TPA (P/3) medium contains per liter: NaCl, 0.5 g; KCl, 8.0 g; NH4Cl, 1.1 g; Tris base, 12.1 g; sodium pyruvate, 0.8 g; 1 ml of 0.16 M Na2SO4, and 0.5% Casamino acids (Difco), pH 7.4. After autoclaving, the following sterile solutions were added: 1 ml of 20% MgCl3 .6H,O, 0.1 ml of 1 M CaCl2, 20 ml of 10% glucose, 25 ml of 0.1 M KH2P04, and 10 ml of thymine (0.2 mg/ml). TPA-low phosphate is TPA (P/3) with the phosphate concentration reduced to 2.5 x 10-' M. Borate buffer is 0.05 M sodium tetraborate. Borate838
PARENTAL RF DNA VOL.VX174 16, 1975 EDTA is 0.05 M sodium tetraborate, 0.006 M EDTA. TE is 0.01 M Tris, 0.001 M EDTA, pH 8.0. Biological assays. Cell density in liquid medium was determined from turbidity measurements in a Bausch and Lomb spectrophotometer at 440 nm in a 1-inch tube against the same medium used as a blank. Preparation of 39P-labeled phage. 32P-labeled 4X174 was prepared as previously described (2). Infection and labeling of cells. E. coli rep3 was diluted 10-fold from an overnight culture (grown without aeration in medium with a fivefold reduced glucose content) into TPA (P/3) and grown in a shaker bath at 37 C to approximately 2 x 101 cells/ml. At this time, the cells were infected with 32P-labeled kX174 am3 at a multiplicity of infection of five. Simultaneously, [H ]thymidine was added to the culture at a concentration of 1 mCi/100 ml of culture. At 7 min after infection, one of two procedures was followed. In the first set of experiments, the culture was chilled for 5 min and then centrifuged in a Sorvall SS-34 rotor at 15,000 rpm for a few seconds in the cold. The pellet was resuspended in a small amount of fresh medium and then further diluted in prewarmed medium plus or minus chloramphenicol (CAM) to a final concentration of 150 Ag/ml. In the second set of experiments, the culture was not centrifuged but was divided and each fraction transferred to a new culture tube containing varying amounts of CAM. At various times, portions of the culture were removed, the cells were lysed, and the DNA was extracted. Cell lysis and DNA extraction. Portions of cells were removed at the appropriate times and added to 0.01 volume of ice-cold 1 M NaCl. The cells were chilled in ice for approximately 5 min and then centrifuged in the cold in a Sorvall centrifuge at 15,000 rpm for a few seconds. The cells were washed three times with 5 ml of borate-EDTA and resuspended in 1 ml of borate-EDTA. Lysozyme was added to a final concentration of 0.4 mg/ml and incubated for 10 min at 37 C. From this point on, any harsh shaking or pipetting of the lysate was avoided to prevent breakage of the bacterial DNA. Sodium dodecyl sulfate was added to a final concentration of 1% and Pronase (predigested at 37 C for 30 min) to a final concentration of 120 gg/ml, and mixed by gentle rolling of the tube. The lysate was incubated at 37 C for an additional 30 min and poured directly onto a high-salt neutral sucrose gradient. The gradient was 5 to 20% sucrose in 1 M NaCl, 0.001 M EDTA, 0.01 M Tris (pH 8), and had a total volume of 34 ml. -Centrifugation was for 17 h at 24,000 rpm in an SW27 rotor at 5 C. Fractions were collected from the top by pumping 50% sucrose into the bottom of the tube through a hypodermic needle. Analysis of DNA on alkaline sucrose gradients. The desired fractions from a neutral sucrose gradient were pooled and ethanol precipitated by adding to each sample 0.1 volume of 3 M sodium acetate and 2 volumes of ice-cold 95% ethanol, and incubating the sample ovemight at -20 C. The samples were then centrifuged in a cold rotor in a Sorvall RC-2 centrifuge at 15,000 rpm for 30 min. The supernatant was poured off and the tubes drained well. Any residual ethanol
839
was removed by attaching the tube to a vacuum line. The precipitate was resuspended in 0.25 ml of TE buffer and 100 gl layered onto a 5 to 20% alkaline sucrose gradient. The gradient contained 5 to 20% sucrose in 0.25 M NaOH and 0.005 M EDTA and was centrifuged for 2 h at 55,000 rpm at 5 C in a SW56 rotor. Eight-drop fractions were collected directly onto Whatman no. 3 filter paper, dried, and counted by liquid scintillation. Centrifugation techniques and radioactivity assay. Centrifugation techniques and liquid scintillation assay of radioactivity have been described in detail previously (1-3).
RESULTS The primary purpose of these experiments was to follow the fate of the infecting parental DNA strand of bacteriophage OX174 upon infection of the Rep, mutant of E. coli. In this host, the parental replicative form is made and the strand-specific nick in the viral strand is introduced but further replication is inhibited (1). The mechanism of inhibition of replication by the rep mutation has yet to be elucidated. The bacteria were infected with virus labeled
with 32P so that the fate of the parental strand of DNA might be observed. DNA replication was followed by the addition of [3H]thymidine at the time of infection. At 7 min after infection, the cells were chilled, centrifuged, and resuspended in fresh medium. A portion was removed for DNA extraction; a second portion was diluted with medium containing no CAM; and a third was diluted to the same cell concentration with medium containing CAM to a final concentration of 150 ,g/ml. Samples were removed at intervals for DNA extraction. The results of 5 to 20% high-salt neutral sucrose velocity sedimentation of lysates of these samples are seen in Fig. 1 and Table 1. Two trends can be observed: first, in the absence of CAM, there is a progressive loss of parental 32P label from the RF I and RF II (open circular RF having one or more single-strand breaks) regions until, by 47 min after infection, very little 39P label remains in the replicative forms. This loss of label is prevented by the addition of CAM. Second, both in the presence and the absence of CAM, there is a progressive shift of label from the RF II to the RF I (21s) regions of the gradient. In earlier work, Francke and Ray (3, 4) observed that most of the molecules sedimenting at 21s in a kX174 infection of rep, were actually in the form of an open double-strand circular DNA containing a single-strand tail of sufficient length to cause the molecule to cosediment with the supercoiled RF I. To test for the
840
J. V IROL.
BOWMAN AND RAY
TABLE 1. Radioactivity in XX replicative forms in E. coli rep8a Source
7min, 17 min, 27min, 47 min, 27min, 47min,
C\J 0 8
CAM CAM CAM CAM + CAM + CAM
-
3H
32P
"2P/'H
72,800 73,700 68,600 28,900 58,000 58,400
19,100 11,400 8,740 3,440 11,700 11,200
0.263 0.154 0.127 0.119 0.202 0.192
aThe total 'H and "P radioactivity in OX replicative forms in the gradients shown in Fig. 1 was determined by summing the aH and "2P radioactivities in all fractions in the RF I and RF II regions of each gradient. The low total radioactivity in the 47-min sample in the absence of CAM was not observed reproducibly and likely resulted from incomplete lysis of that particular sample.
presence of such molecules in the present experiments, the 21s fractions were pooled, ethanol 4 precipitated, and sedimented on 5 to 20% highsalt alkaline sucrose velocity gradients. Under denaturing conditions, such as these, true RF I 0 will sediment very rapidly, much more so than a (f)47min+CAM (e) 27 min + CAM nicked molecule which will sediment as circular and linear single-stranded DNA. The results are 16seen in Fig. 2. In the absence of CAM, there is an increase with time in the proportion of DNA sedimenting as single-stranded DNA (fraction 1218 to 20) and a concomitant decrease in denatured supercoils (fractions 7 to 9). On the other hand, where CAM is present, the majority of the DNA sediments in alkali as denatured supercoils. To examine the possibility that viral strand degradation might result from an abortive attempt at single-strand synthesis (RF _ SS), we have examined the concentration dependence of CAM inhibition of viral strand degradation. A 10 20 30 40 0 10 20 30 40 CAM concentration of 30 ,g/ml is sufficient to FRACTION prevent RF - SS synthesis but not RF - RF FIG. 1. Neutral sucrose velocity sedimentation of replication in a wild-type host (9, 10). Sufficient parental RF formed upon infection of Rep- cells with gene A protein is synthesized at that concentraqOX174 am3 with and without CAM at 150 jg/ml. E. its subsecoli rep. cells were infected with 32P-labeled 4X174 tion to allow nicking of the RF and am3 at a multiplicity of infection of five. [3H]thymi- quent replication. At a CAM concentration of dine was added at the time of infection. Seven 150 ug/ml, no gene A protein is synthesized and minutes later, the culture was divided and 150 Ag of both RF - SS and RF _ RF replication are CAM/ml added to one part. Aliquots (15 ml) were inhibited. removed at the times indicated, the DNA extracted, Infection and extraction of the DNA was done and sedimented on a neutral sucrose gradient. (a) as in Fig. 1, except that after the initial 7 min of DNA extracted 7 min after infection; (b) DNA ex- infection the culture was divided into four parts tracted 17 min after infection; (c) DNA extracted 27 and different amounts of CAM added to each to min after infection; (d) DNA extracted 47 min after give final concentrations of 0, 10, 30, and 150 ;g infection; (e) CAM added at 7 min after infection and of incubamin After 40 additional of CAM/ml. 7 at added min CAM DNA extracted 20 min later; (f) after infection and DNA extracted 40 min later. tion, a time by which all of the parental label would be degraded in the absence of CAM (Fig. Sedimentation is from left to right. "2p, 0; 'H, 0.
V 1X174 VOL. 16, 1975
PARENTAL RF DNA
841
1), the DNA was extracted as before and sedimented on a 5 to 20% high-salt neutral sucrose gradient. The results are shown in Fig. 3. Two things are apparent from these graphs. First, 8 the degradation of the parental label is only significantly inhibited by 150 ug of CAM/ml. Second, different amounts of CAM have an 6 effect on the proportion of label in each peak. At CAM concentrations of 10 and 30 gg/ml, there is a much greater proportion of label in the RF II 4 peak than when the concentration of CAM is I1+teither 0 or 150 ,ug/ml. i \ z To further elucidate the nature of the mole2 cules sedimenting in the 21s regions, these fractions of each gradient were pooled, the DNA ___ _, / > ethanol precipitated and run on a 5 to 20% (d)47min-CAM (c) 27min-CAM high-salt alkaline sucrose velocity gradient. The results are presented in Fig. 4. As the concentra18 tion of CAM is increased from 0 to 150 jg/ml, .1.1 the fraction of the 21s DNA sedimenting as K) denatured supercoils increases until at 150 jig/ml essentially all the label appears in this C and elongation of peak. Thus, botharedegradation 08 inhibited at a CAM concenthe viral strand x tration of 150 Ag/ml. Concentrations of 10 or 30 -g/ml significantly reduce the amount of RF cl6 T o with elongated tails but do not prevent viral strand degradation. (a) 7min- CAM
0-
(b)17min-CAM
X
4-
2
2-}
(e) 27min+ CAM 16 12 8
8
4
O
o10
20 20
DISCUSSION these From results, it appears that two proc} lesses are occurring in the replicative form DNA A of 4X174 synthesized in a Rep- host. First, there is a progressive conversion of replicative intermediates from RF II to species sedimenting (f) 47min+ CAM at 21s. This results from an elongation of one of the DNA strands of an RF II molecule rather than by sealing of a nick or gap (3). Secondly, the parental viral strand is progressively degraded so that little parental label remains by 47 min after infection. A possible mechanism by which the viral strand might be degraded is by nick translation, a process whereby DNA is degraded at the 5' terminus of a nick in a duplex DNA while nucleotides are added at the 3' terminus (7). It is known that the initial nick in 4X174 replicative DNA is in the parental viral strand (3, 4). Were nick translation to occur, the parental ~ 110l 20 FRACTION
FiG. 2. Alkaline sucrose velocity sedimentation of the RF I regions of Fig. 1. The 21s fractions from each gradient in Fig. 1 were combined, ethanol precipitated, and sedimented on an alkaline sucrose velocity gradient. (a) Fractions 22 to 24 from Fig. Ia; (b) fractions 23 to 25 from Fig. Ib; (c) fractions 22 to 25
DNA would be gradually degraded and replaced
by newly synthesized DNA. Our observations from Fig. Ic; (d) fractions 23 to 25 from Fig. Id; (e) fractions 22 to 24 from Fig. le; (f) fractions 24 to 26 from Fig. If. The vertical arrows indicate the position of denatured supercoils. Sedimentation is from right to left. 32p, 0; 3H, *.
842
BOWMAN AND RAY
J. VIROL.
(b) + IOpg/ml CAM
(a)-CAM N C\J
I C
/ (c)+30pg/mI CAM (d) + 150pg/mICAM
0
XE (-9
I
C
O0
10
-p~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 20 30 40 0 10 20 30 40
FRACTION FIG. 3. Neutral sucrose velocity sedimentation of parental RF formed upon infection of Rep-cells with OX174 am3 in the presence of varying amounts of CAM. A 60-ml culture of E. coli rep, was infected with 32P-labeled OX174 at a multiplicity of infection of five. [3H]thymidine was added at the time of infection. At 7 min after infection the culture was divided into four equal parts and CAM was added at various concentrations. Forty minutes later the DNA was extracted and sedimented on a neutral sucrose velocity gradient. (a) No CAM; (b) 10 ug of CAM/ml; (c) 30 Ag of CAM/ml; (d) 150 ,ug of CAM/ml. Sedimentation is from left to right. S2p 0; 3HO are consistent with such a mechanism but do not exclude other alternatives. The addition of CAM had a profound effect
the degradation of parental label. CAM is an inhibitor of protein synthesis in bacteria and, at the level of 150 Ag/ml used in the initial experiment, would inhibit both RF SS and
on
RF
RF
synthesis
in
a
wild-type host. The
addition of CAM at 150 ,g/ml as late as 7 min after infection prevents the loss of parental label although it shifts from the RF II to the RF I peak. The shift to the 21s position in this case appears to be due almost entirely to the closure of the nick in the RF II and not to elongation of the parental strand. Perhaps by preventing the further synthesis of the gene A endonuclease, which appears to act only once (6), CAM allows the nick to be repaired by host enzymes, thus protecting the DNA from degradation. It was of interest to find out whether preventing the synthesis of proteins necessary for single-strand synthesis would effectively eliminp+^
the formation of the elongated tails and the degradation of the parental label. A time was chosen at which point almost all of the parental label is degraded in the absence of CAM. Concentrations of 10 and 30,ug of CAM/ml did not prevent the degradation of the parental label but did inhibit the conversion of much of the material to denaturable 21s structures. These results suggest that viral strand degradation does not require elongation of the viral strand and displacement of a single-stranded tail. From the total inhibition of both viral strand degradation and elongation by CAM at 150 Ag/ml, a concentration which prevents gene A expression, we infer that the gene A-specific nicking of the parental RF is necessary for the subsequent degradation of the viral strand. ACKNOWLEDGMENTS This research was supported by a Public Health Service grant AI-10752 from the National Institute of Allergy and Infectious Diseases. K. L. B. was supported as a predoctoral trainee on a Public Health Service training grant GM 1531
VOL.VX174 16, 1975 PARENTAL RF DNA (a)-CAM (b)++ IOpg/mI (a)-CAM(b) 10 pg /m CAM CAM 6 4_
2
c
.
(c) ..3Oupg/ml CAM
(d) +150 pg/mI CAM
from the National Institute of General Medical Sciences to
the Molecular Biology Institute.
LITERATURE CITED 1. Denhardt, D. J., D. H. Dressler, and A. Hathaway. 1967. The abortive replication of OX174 DNA in a recombination-deficient mutant of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 57:813-820. 2. Francke, B., and D. S. Ray. 1971. Fate of parental OX174 DNA upon infection of starved thymine-requiring host cells. Virology 4:168-187. 3. Francke, B., and D. S. Ray. 1971. Formation of the parental replicative form DNA of bacteriophage OX174 and initial events in its replication. J. Mol. Biol. 4.
CY
o10
l5.
L_
08
6.
6_ 7.
4
8.
2OO_6z W 10
9.
w 20
0
-
f c0
f
20
843
10.
61:565-586. Francke, B., and D. S. Ray. 1972. Cis-limited action of
the gene A product of bacteriophage OX174 and the essential bacterial site. Proc. Natl. Acad. Sci. U. S. A. 69:475-479. Gilbert, W., and D. H. Dressler. 1968. DNA replication: the rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 33:473-484. Henry, T. J., and R. Knippers. 1974. Isolation and function of the gene A initiator of bacteriophage 4X174, a highly specific RNA endonuclease. Proc. Natl. Acad. Sci. U. S. A. 71:1549-1553. Kornberg, A. 1969. Active center of DNA polymerase. Science 163:1410-1418. Schroder, C. H., and H. C. Kaerner. 1972. Replication of bacteriophage OX174 replicative form DNA in vivo. J. Mol. Biol. 71:351-362. Sinsheimer, R. L. 1968. Bacteriophage OX174 and related
viruses, Prog. Nucl. Acid Res. Mol. Biol. 8:115-169.
Tessman, E. 1966. Mutants of bacteriophage S13 blocked in infectious DNA synthesis. J. Mol. Biol. 17:218-236.
FRACTION FIG. 4. Alkaline sucrose velocity sedimentation of the RF I regions of Fig. 3. The 21s fractions from each gradient in Fig. 3 were combined, ethanol precipitated, and sedimented on an alkaline sucrose velocity gradient. (a) Fraction 25 to 27 from Fig. 3a;
(b) fractions 26 to 28 from Fig. 3b; (c) fractions 26 to 28 from Fig. 3c; (d) fractions 26 to 28 from Fig. 3d. The vertical arrows indicate the position of denatured supercoils. Sedimentation is from right to left. S2p, 0; 3H, .
JOURNAL OF VIROLOGY, Oct. 1975, p. 838-843 Copyright 0 1975 American Society for Microbiology
Degradation of the Viral Strand of OX174 Parental Replicative-Form DNA in a Rep- Host KATHERINE L. BOWMAN AND DAN S. RAY* Molecular Biology Institute and Department of Biology, University of California, Los Angeles, California 90024 Received for publication 6 May 1975
A progressive degradation of the parental viral strand label is observed upon infection of a Rep- mutant of Escherichia coli by 32P-labeled OX174. Very little parental label remains in the RF (replicative form) by 47 min after infection. Concomitant with this degradation, replicative intermediates are formed which sediment at 21s, the rate of RF I (supercoiled-closed circular DNA), in a neutral sucrose gradient but which denature and sediment in alkaline gradients as single strands of unit size and larger. These denaturable 21s replicative intermediates have been shown previously to be RF molecules containing an elongated viral strand. Addition of chloramphenicol at 7 min after infection at 30 gg/ml, a concentration sufficient to block RF
SS (single strand) synthesis but not RF
-
RF synthesis in a wild-type host cell, reduced the amount of viral strand elongation but did not prevent viral strand degradation. The addition of chloramphenicol at 150,ug/ml at 7 min after infection totally prevents both the degradation of the parental label and the formation of the replicative intermediates with elongated tails. We infer that degradation of the viral strand requires the gene A-mediated nicking of the viral strand but not the concomitant elongation of the viral strand.
The replicative cycle of 4X174 DNA can be divided into three stages: (i) conversion of the parental single-stranded circular DNA to the double-stranded circular replicative form (SS - RF), the initial copy of which is termed the parental replicative form; (ii) RF replication (RF -- RF) in which numerous copies of the RF are made; and (iii) asymmetric synthesis of progeny single-stranded DNA (RF - SS) (5, 8,9). Only a single viral gene product, the gene A protein, is involved in RF - RF synthesis (10). Replication of the parental RF depends on a specific endonucleolytic cleavage of the viral strand of the RF by the OX174 gene A protein (6). In the absence of a functional gene A protein, the parental RF accumulates as RF I and does not replicate further (3). The first stage of replication, SS - RF occurs normally in a Rep- host and is followed by covalent closure of the complementary strand. In the presence of a wild-type gene A, this parental RF molecule is nicked in the viral strand (3). Further replication is blocked in a Rep- host. However, upon prolonged incubation of infected Rep- cells, a small amount of viral strand synthesis occurs by extension of the nicked viral strand (3, 4). This synthesis beyond the block imposed by the Rep - mutation leads
to the formation of intermediate structures sedimenting in neutral gradients at 21s, the same rate as the supercoiled RF I. Yet, upon alkaline sedimentation, these structures yield circular complementary strands and viral strands of greater than unit length. By electron microscopy, these intermediates appear to have a sigma structure, a relaxed duplex ring with a single-stranded tail (4). In this paper, the formation of these 21s denaturable replicative intermediates is discussed with an emphasis on the fate of the viral strand in the presence of a wild-type gene A.
MATERIALS AND METHODS Phage and bacterial strains. The sources and properties of the strains used here have been described previously (2, 3). The Rep- mutant used was Escherichia coli rep, (1). Media and solutions. TPA (P/3) medium contains per liter: NaCl, 0.5 g; KCl, 8.0 g; NH4Cl, 1.1 g; Tris base, 12.1 g; sodium pyruvate, 0.8 g; 1 ml of 0.16 M Na2SO4, and 0.5% Casamino acids (Difco), pH 7.4. After autoclaving, the following sterile solutions were added: 1 ml of 20% MgCl3 .6H,O, 0.1 ml of 1 M CaCl2, 20 ml of 10% glucose, 25 ml of 0.1 M KH2P04, and 10 ml of thymine (0.2 mg/ml). TPA-low phosphate is TPA (P/3) with the phosphate concentration reduced to 2.5 x 10-' M. Borate buffer is 0.05 M sodium tetraborate. Borate838
PARENTAL RF DNA VOL.VX174 16, 1975 EDTA is 0.05 M sodium tetraborate, 0.006 M EDTA. TE is 0.01 M Tris, 0.001 M EDTA, pH 8.0. Biological assays. Cell density in liquid medium was determined from turbidity measurements in a Bausch and Lomb spectrophotometer at 440 nm in a 1-inch tube against the same medium used as a blank. Preparation of 39P-labeled phage. 32P-labeled 4X174 was prepared as previously described (2). Infection and labeling of cells. E. coli rep3 was diluted 10-fold from an overnight culture (grown without aeration in medium with a fivefold reduced glucose content) into TPA (P/3) and grown in a shaker bath at 37 C to approximately 2 x 101 cells/ml. At this time, the cells were infected with 32P-labeled kX174 am3 at a multiplicity of infection of five. Simultaneously, [H ]thymidine was added to the culture at a concentration of 1 mCi/100 ml of culture. At 7 min after infection, one of two procedures was followed. In the first set of experiments, the culture was chilled for 5 min and then centrifuged in a Sorvall SS-34 rotor at 15,000 rpm for a few seconds in the cold. The pellet was resuspended in a small amount of fresh medium and then further diluted in prewarmed medium plus or minus chloramphenicol (CAM) to a final concentration of 150 Ag/ml. In the second set of experiments, the culture was not centrifuged but was divided and each fraction transferred to a new culture tube containing varying amounts of CAM. At various times, portions of the culture were removed, the cells were lysed, and the DNA was extracted. Cell lysis and DNA extraction. Portions of cells were removed at the appropriate times and added to 0.01 volume of ice-cold 1 M NaCl. The cells were chilled in ice for approximately 5 min and then centrifuged in the cold in a Sorvall centrifuge at 15,000 rpm for a few seconds. The cells were washed three times with 5 ml of borate-EDTA and resuspended in 1 ml of borate-EDTA. Lysozyme was added to a final concentration of 0.4 mg/ml and incubated for 10 min at 37 C. From this point on, any harsh shaking or pipetting of the lysate was avoided to prevent breakage of the bacterial DNA. Sodium dodecyl sulfate was added to a final concentration of 1% and Pronase (predigested at 37 C for 30 min) to a final concentration of 120 gg/ml, and mixed by gentle rolling of the tube. The lysate was incubated at 37 C for an additional 30 min and poured directly onto a high-salt neutral sucrose gradient. The gradient was 5 to 20% sucrose in 1 M NaCl, 0.001 M EDTA, 0.01 M Tris (pH 8), and had a total volume of 34 ml. -Centrifugation was for 17 h at 24,000 rpm in an SW27 rotor at 5 C. Fractions were collected from the top by pumping 50% sucrose into the bottom of the tube through a hypodermic needle. Analysis of DNA on alkaline sucrose gradients. The desired fractions from a neutral sucrose gradient were pooled and ethanol precipitated by adding to each sample 0.1 volume of 3 M sodium acetate and 2 volumes of ice-cold 95% ethanol, and incubating the sample ovemight at -20 C. The samples were then centrifuged in a cold rotor in a Sorvall RC-2 centrifuge at 15,000 rpm for 30 min. The supernatant was poured off and the tubes drained well. Any residual ethanol
839
was removed by attaching the tube to a vacuum line. The precipitate was resuspended in 0.25 ml of TE buffer and 100 gl layered onto a 5 to 20% alkaline sucrose gradient. The gradient contained 5 to 20% sucrose in 0.25 M NaOH and 0.005 M EDTA and was centrifuged for 2 h at 55,000 rpm at 5 C in a SW56 rotor. Eight-drop fractions were collected directly onto Whatman no. 3 filter paper, dried, and counted by liquid scintillation. Centrifugation techniques and radioactivity assay. Centrifugation techniques and liquid scintillation assay of radioactivity have been described in detail previously (1-3).
RESULTS The primary purpose of these experiments was to follow the fate of the infecting parental DNA strand of bacteriophage OX174 upon infection of the Rep, mutant of E. coli. In this host, the parental replicative form is made and the strand-specific nick in the viral strand is introduced but further replication is inhibited (1). The mechanism of inhibition of replication by the rep mutation has yet to be elucidated. The bacteria were infected with virus labeled
with 32P so that the fate of the parental strand of DNA might be observed. DNA replication was followed by the addition of [3H]thymidine at the time of infection. At 7 min after infection, the cells were chilled, centrifuged, and resuspended in fresh medium. A portion was removed for DNA extraction; a second portion was diluted with medium containing no CAM; and a third was diluted to the same cell concentration with medium containing CAM to a final concentration of 150 ,g/ml. Samples were removed at intervals for DNA extraction. The results of 5 to 20% high-salt neutral sucrose velocity sedimentation of lysates of these samples are seen in Fig. 1 and Table 1. Two trends can be observed: first, in the absence of CAM, there is a progressive loss of parental 32P label from the RF I and RF II (open circular RF having one or more single-strand breaks) regions until, by 47 min after infection, very little 39P label remains in the replicative forms. This loss of label is prevented by the addition of CAM. Second, both in the presence and the absence of CAM, there is a progressive shift of label from the RF II to the RF I (21s) regions of the gradient. In earlier work, Francke and Ray (3, 4) observed that most of the molecules sedimenting at 21s in a kX174 infection of rep, were actually in the form of an open double-strand circular DNA containing a single-strand tail of sufficient length to cause the molecule to cosediment with the supercoiled RF I. To test for the
840
J. V IROL.
BOWMAN AND RAY
TABLE 1. Radioactivity in XX replicative forms in E. coli rep8a Source
7min, 17 min, 27min, 47 min, 27min, 47min,
C\J 0 8
CAM CAM CAM CAM + CAM + CAM
-
3H
32P
"2P/'H
72,800 73,700 68,600 28,900 58,000 58,400
19,100 11,400 8,740 3,440 11,700 11,200
0.263 0.154 0.127 0.119 0.202 0.192
aThe total 'H and "P radioactivity in OX replicative forms in the gradients shown in Fig. 1 was determined by summing the aH and "2P radioactivities in all fractions in the RF I and RF II regions of each gradient. The low total radioactivity in the 47-min sample in the absence of CAM was not observed reproducibly and likely resulted from incomplete lysis of that particular sample.
presence of such molecules in the present experiments, the 21s fractions were pooled, ethanol 4 precipitated, and sedimented on 5 to 20% highsalt alkaline sucrose velocity gradients. Under denaturing conditions, such as these, true RF I 0 will sediment very rapidly, much more so than a (f)47min+CAM (e) 27 min + CAM nicked molecule which will sediment as circular and linear single-stranded DNA. The results are 16seen in Fig. 2. In the absence of CAM, there is an increase with time in the proportion of DNA sedimenting as single-stranded DNA (fraction 1218 to 20) and a concomitant decrease in denatured supercoils (fractions 7 to 9). On the other hand, where CAM is present, the majority of the DNA sediments in alkali as denatured supercoils. To examine the possibility that viral strand degradation might result from an abortive attempt at single-strand synthesis (RF _ SS), we have examined the concentration dependence of CAM inhibition of viral strand degradation. A 10 20 30 40 0 10 20 30 40 CAM concentration of 30 ,g/ml is sufficient to FRACTION prevent RF - SS synthesis but not RF - RF FIG. 1. Neutral sucrose velocity sedimentation of replication in a wild-type host (9, 10). Sufficient parental RF formed upon infection of Rep- cells with gene A protein is synthesized at that concentraqOX174 am3 with and without CAM at 150 jg/ml. E. its subsecoli rep. cells were infected with 32P-labeled 4X174 tion to allow nicking of the RF and am3 at a multiplicity of infection of five. [3H]thymi- quent replication. At a CAM concentration of dine was added at the time of infection. Seven 150 ug/ml, no gene A protein is synthesized and minutes later, the culture was divided and 150 Ag of both RF - SS and RF _ RF replication are CAM/ml added to one part. Aliquots (15 ml) were inhibited. removed at the times indicated, the DNA extracted, Infection and extraction of the DNA was done and sedimented on a neutral sucrose gradient. (a) as in Fig. 1, except that after the initial 7 min of DNA extracted 7 min after infection; (b) DNA ex- infection the culture was divided into four parts tracted 17 min after infection; (c) DNA extracted 27 and different amounts of CAM added to each to min after infection; (d) DNA extracted 47 min after give final concentrations of 0, 10, 30, and 150 ;g infection; (e) CAM added at 7 min after infection and of incubamin After 40 additional of CAM/ml. 7 at added min CAM DNA extracted 20 min later; (f) after infection and DNA extracted 40 min later. tion, a time by which all of the parental label would be degraded in the absence of CAM (Fig. Sedimentation is from left to right. "2p, 0; 'H, 0.
V 1X174 VOL. 16, 1975
PARENTAL RF DNA
841
1), the DNA was extracted as before and sedimented on a 5 to 20% high-salt neutral sucrose gradient. The results are shown in Fig. 3. Two things are apparent from these graphs. First, 8 the degradation of the parental label is only significantly inhibited by 150 ug of CAM/ml. Second, different amounts of CAM have an 6 effect on the proportion of label in each peak. At CAM concentrations of 10 and 30 gg/ml, there is a much greater proportion of label in the RF II 4 peak than when the concentration of CAM is I1+teither 0 or 150 ,ug/ml. i \ z To further elucidate the nature of the mole2 cules sedimenting in the 21s regions, these fractions of each gradient were pooled, the DNA ___ _, / > ethanol precipitated and run on a 5 to 20% (d)47min-CAM (c) 27min-CAM high-salt alkaline sucrose velocity gradient. The results are presented in Fig. 4. As the concentra18 tion of CAM is increased from 0 to 150 jg/ml, .1.1 the fraction of the 21s DNA sedimenting as K) denatured supercoils increases until at 150 jig/ml essentially all the label appears in this C and elongation of peak. Thus, botharedegradation 08 inhibited at a CAM concenthe viral strand x tration of 150 Ag/ml. Concentrations of 10 or 30 -g/ml significantly reduce the amount of RF cl6 T o with elongated tails but do not prevent viral strand degradation. (a) 7min- CAM
0-
(b)17min-CAM
X
4-
2
2-}
(e) 27min+ CAM 16 12 8
8
4
O
o10
20 20
DISCUSSION these From results, it appears that two proc} lesses are occurring in the replicative form DNA A of 4X174 synthesized in a Rep- host. First, there is a progressive conversion of replicative intermediates from RF II to species sedimenting (f) 47min+ CAM at 21s. This results from an elongation of one of the DNA strands of an RF II molecule rather than by sealing of a nick or gap (3). Secondly, the parental viral strand is progressively degraded so that little parental label remains by 47 min after infection. A possible mechanism by which the viral strand might be degraded is by nick translation, a process whereby DNA is degraded at the 5' terminus of a nick in a duplex DNA while nucleotides are added at the 3' terminus (7). It is known that the initial nick in 4X174 replicative DNA is in the parental viral strand (3, 4). Were nick translation to occur, the parental ~ 110l 20 FRACTION
FiG. 2. Alkaline sucrose velocity sedimentation of the RF I regions of Fig. 1. The 21s fractions from each gradient in Fig. 1 were combined, ethanol precipitated, and sedimented on an alkaline sucrose velocity gradient. (a) Fractions 22 to 24 from Fig. Ia; (b) fractions 23 to 25 from Fig. Ib; (c) fractions 22 to 25
DNA would be gradually degraded and replaced
by newly synthesized DNA. Our observations from Fig. Ic; (d) fractions 23 to 25 from Fig. Id; (e) fractions 22 to 24 from Fig. le; (f) fractions 24 to 26 from Fig. If. The vertical arrows indicate the position of denatured supercoils. Sedimentation is from right to left. 32p, 0; 3H, *.
842
BOWMAN AND RAY
J. VIROL.
(b) + IOpg/ml CAM
(a)-CAM N C\J
I C
/ (c)+30pg/mI CAM (d) + 150pg/mICAM
0
XE (-9
I
C
O0
10
-p~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 20 30 40 0 10 20 30 40
FRACTION FIG. 3. Neutral sucrose velocity sedimentation of parental RF formed upon infection of Rep-cells with OX174 am3 in the presence of varying amounts of CAM. A 60-ml culture of E. coli rep, was infected with 32P-labeled OX174 at a multiplicity of infection of five. [3H]thymidine was added at the time of infection. At 7 min after infection the culture was divided into four equal parts and CAM was added at various concentrations. Forty minutes later the DNA was extracted and sedimented on a neutral sucrose velocity gradient. (a) No CAM; (b) 10 ug of CAM/ml; (c) 30 Ag of CAM/ml; (d) 150 ,ug of CAM/ml. Sedimentation is from left to right. S2p 0; 3HO are consistent with such a mechanism but do not exclude other alternatives. The addition of CAM had a profound effect
the degradation of parental label. CAM is an inhibitor of protein synthesis in bacteria and, at the level of 150 Ag/ml used in the initial experiment, would inhibit both RF SS and
on
RF
RF
synthesis
in
a
wild-type host. The
addition of CAM at 150 ,g/ml as late as 7 min after infection prevents the loss of parental label although it shifts from the RF II to the RF I peak. The shift to the 21s position in this case appears to be due almost entirely to the closure of the nick in the RF II and not to elongation of the parental strand. Perhaps by preventing the further synthesis of the gene A endonuclease, which appears to act only once (6), CAM allows the nick to be repaired by host enzymes, thus protecting the DNA from degradation. It was of interest to find out whether preventing the synthesis of proteins necessary for single-strand synthesis would effectively eliminp+^
the formation of the elongated tails and the degradation of the parental label. A time was chosen at which point almost all of the parental label is degraded in the absence of CAM. Concentrations of 10 and 30,ug of CAM/ml did not prevent the degradation of the parental label but did inhibit the conversion of much of the material to denaturable 21s structures. These results suggest that viral strand degradation does not require elongation of the viral strand and displacement of a single-stranded tail. From the total inhibition of both viral strand degradation and elongation by CAM at 150 Ag/ml, a concentration which prevents gene A expression, we infer that the gene A-specific nicking of the parental RF is necessary for the subsequent degradation of the viral strand. ACKNOWLEDGMENTS This research was supported by a Public Health Service grant AI-10752 from the National Institute of Allergy and Infectious Diseases. K. L. B. was supported as a predoctoral trainee on a Public Health Service training grant GM 1531
VOL.VX174 16, 1975 PARENTAL RF DNA (a)-CAM (b)++ IOpg/mI (a)-CAM(b) 10 pg /m CAM CAM 6 4_
2
c
.
(c) ..3Oupg/ml CAM
(d) +150 pg/mI CAM
from the National Institute of General Medical Sciences to
the Molecular Biology Institute.
LITERATURE CITED 1. Denhardt, D. J., D. H. Dressler, and A. Hathaway. 1967. The abortive replication of OX174 DNA in a recombination-deficient mutant of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 57:813-820. 2. Francke, B., and D. S. Ray. 1971. Fate of parental OX174 DNA upon infection of starved thymine-requiring host cells. Virology 4:168-187. 3. Francke, B., and D. S. Ray. 1971. Formation of the parental replicative form DNA of bacteriophage OX174 and initial events in its replication. J. Mol. Biol. 4.
CY
o10
l5.
L_
08
6.
6_ 7.
4
8.
2OO_6z W 10
9.
w 20
0
-
f c0
f
20
843
10.
61:565-586. Francke, B., and D. S. Ray. 1972. Cis-limited action of
the gene A product of bacteriophage OX174 and the essential bacterial site. Proc. Natl. Acad. Sci. U. S. A. 69:475-479. Gilbert, W., and D. H. Dressler. 1968. DNA replication: the rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 33:473-484. Henry, T. J., and R. Knippers. 1974. Isolation and function of the gene A initiator of bacteriophage 4X174, a highly specific RNA endonuclease. Proc. Natl. Acad. Sci. U. S. A. 71:1549-1553. Kornberg, A. 1969. Active center of DNA polymerase. Science 163:1410-1418. Schroder, C. H., and H. C. Kaerner. 1972. Replication of bacteriophage OX174 replicative form DNA in vivo. J. Mol. Biol. 71:351-362. Sinsheimer, R. L. 1968. Bacteriophage OX174 and related
viruses, Prog. Nucl. Acid Res. Mol. Biol. 8:115-169.
Tessman, E. 1966. Mutants of bacteriophage S13 blocked in infectious DNA synthesis. J. Mol. Biol. 17:218-236.
FRACTION FIG. 4. Alkaline sucrose velocity sedimentation of the RF I regions of Fig. 3. The 21s fractions from each gradient in Fig. 3 were combined, ethanol precipitated, and sedimented on an alkaline sucrose velocity gradient. (a) Fraction 25 to 27 from Fig. 3a;
(b) fractions 26 to 28 from Fig. 3b; (c) fractions 26 to 28 from Fig. 3c; (d) fractions 26 to 28 from Fig. 3d. The vertical arrows indicate the position of denatured supercoils. Sedimentation is from right to left. S2p, 0; 3H, .
