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Arrest of host DNA synthesis in Bacillus subtilis infected with phage ∅e
VIROLOQY
49,
668-674 (1972)
Arrest
of Host
DNA
Synthesis with
URl Department
LAVI
AND
of Genetics,
in Bacillus
Phage
Infected
4e
MENASHE
The Hebrew
sobtilis
MARCUS
University,
Jerusalem,
Israel
Accepted May 26, 19% Arrest of host DNA synthesis in Bacillus subtilis infected with the virulent phage be is dependent on the synthesis of a phage-coded protein (Marcus and Newlon, 1971). The mechanism of host DNA arrest was studied in toluene-treated B. subtilis cells. The results showed that preparations of toluenized phage infected cells manifest faithfully the kinetics and specificity of host DNA arrest and the appearance of phage DNA synthesis. The arrest of host DNA synthesis is not caused by the production in the host DNA of single strand breaks that can be repaired by the DNA polymerase repair enzyme (Pol I). Neither hydroxymethyl deoxyuridylate (HMdUMP) nor probably any other small molecule is responsible for host DNA arrest. INTRODUCTION
Bacteriophage t$e, which infects Bacillus subtilis, arrests host DNA synthesis a few minutes after infection (Roscoe, 196913; Marcus and Newlon, 1971). This virulent phage contains hydroxymethyluracil (HMU) instead of thymine in its DNA, and induces the synthesis of several proteins, including an inhibitor of the bacterial thymidylate synthetase and a deoxythymidine nucleotidohydrolase (dTTPase) early after infection (Roscoe and Tucker, 1966; Roscoe, 1969a). The arrest of host DNA synthesis in the infected cells was shown not to be dependent on the depletion of the dTTP pool in the infected cells due to the action of thymidylate synthetase inhibitor or dTTPase. Inhibition occurred in thymine requiring bacterial mutants infected with dTTPaseless phage mutants in the presence of exogenous thymine (Roscoe, 1969b; Marcus and Newlon, 1971). Depletion of the dTTP pool in the infected cells is probably important for the normal growth cycle of the phage. Phage mutants that do not destroy the dTTP pool in the infected cells incorporate thymine into DNA (Roscoe, 1969b; Marcus and Newlon, 1971). Replacement of HMU by thymine 668 Copyright @ ltl72 by Academic Press, Inc. All rights of reproduction in any form reserved.
above a critical level interferes with phage DNA synthesis (Marcus, unpublished resuits) . Further investigation on the mechanism of host DNA arrest was based on studies of DNA synthesis in toluene treated cells of B. subtilis wild-type strain and its DNA polymerase negative mutant (Pol I-). The results discussed in this paper show that: (1) The kinetics of host and phage DNA synthesis in toluene treated cells are similar to those obtained in vivo. (2) The absence of hydroxymethyl deoxyuridylate (HMdUMP) or its derivatives in cells infect’ed with phage does not prevent host DNA arrest. (3) Host DNA arrest is not caused by the production of repairable single strand breaks in the host chromosome. MATERIALS
AND
METHODS
Microorganisms. The bacterial strains are derivatives of B. subtilis 168. The phages are derivatives of 4e. A list of the strains is presented in Table 1. Media. LB Broth (Marcus and Newlon, 1971) was used throughout this work. Assay of DlVA synthesis in toluene treated uninfected and, infected cells. Bacteria were grown in LB broth to Fi X 107 cells/ml.
TABLE
PHAGE AND
Bacteria 168 168 Phage +e S27 bda37 (derived from 527) 5/18/R al (derived from
OF HOST
STRAINS
USED~
1
BACTERIAL
Strain
ARREST
Relevant genotype
Wild type PO1 IWild type sus DNA-negative dTTPase-n HMsus DNA-newtive dTTPaseHMdTTPasedTTPaseDNAtS
bda-
5/18/R) a Abbreviations: Pol I, DNA polymerase (repair enzyme) ; dTTPase, deoxythymidine triphosphate nucleotidohydrolase; HM, deoxyuridylate hydroxymethylase; bda, bacterial DNA arrest.
DNA
SYNTHESIS
669
filtered on Whatman GF/C filters, washed with 15 ml of ice-cold 1% TCA, dried and counted in a 3380 Packard Liquid Scintillation Spectrometer. Density gradient centrifugation. CsCl equilibrium gradients were performed as described by Roscoe (1969b). CsCl (99.9%) was purchased from Varlacoid Chemical Company. Centrifugation was carried out in a L2-50 Beckman Preparative Ultracentrifuge with a SW 50.1 rotor for 48 hr at 35,000 rpm at 22”. RESULTS
In a previous paper (Marcus and Newlon, 1971) the possibility that the arrest of host DNA synthesis by phage $e resulted from inhibition of the host replication complex by the phage was explored. Activity of DNA polymerase was determined in crude and partially purified extracts prepared according to Ganesan (1968). Specific activities of DNA polymerase in extracts of uninfected cells and cells infected with the DNA-negative phage mutant S27 were similarly high and probably represented the activity of a repair enzyme (Marcus and Newlon, 1971; De Lucia and Cairns, 1969). It has since been found that DNA replication can be studied in toluenetreated cells of E. coli (Moses and Richardson, 1970; Mordoh et al., 1970; Kohiyama and Kolber, 1970; Burger, 1971) and B. subt&s (Matsushita et al., 1970; Billen et al., 1971). We decided to use a pal I- mutant of B. subtilis in order to eliminate the possibility of repair polymerase activity which occurs in toluene-treated cells under certain conditions (Moses and Richardson, 1970). Dr. J. Gross kindly supplied us with the B. subtilis pal Imutant. DNA synthesis in this mutant after toluene treatment was found to be dependent on the presence of ATP and was inhibited by p-chloromercuribenzoate.
Cell density was determined by direct counting in a Petroff-Hauser cell. Phage infection was carried out at this cell density at a multiplicity of infection (m.0.i) of 10. In all experiments, survival of cells 10 min after infection was l-5%. At various times, 15-ml aliquots were washed and suspended in 1 ml of phosphate buffer 0.05 M pH 7.4. Toluene (10 ~1) was added and the suspension was vigorously agitated on a Vortex mixer for 1 min at room temperature. The reaction mixture was based on that of Kohiyama and Kolber (1970) with some modifications: 0.05 M Tris buffer pH 7.4; 0.005 M MgC12; 0.09 M KCI; 0.001 M dithiothreitol; 1 X lop3 M ATP; 3 X lo+ M of: dATP, dCTP, dGTP; 7 X 10-e M dTTP; 0.15 #Zi 3H-dTTP; 0.001 M NaN3; 3.5-7 X lo7 cells. The cells were added to the mixture immediately after toluene treatment. Water was added to a final volume of 0.15 ml. The reaction was terminated after 30 min at the specified temperature by addition of Host DNA Synthesis in Cells Infected with 0.15 ml of 1 M NaOH and 0.02 M ethylenePhage Mutants 827 and bdaS7 diaminetetraacetic acid. The mixture was Host DNA synthesis in vivo at 37” is heated for 30 set at loo”, cooled immearrested at about 14 min after infection diately to 0” and the following additions phage mutant were made: 1 M HCI 0.15 ml, 0.1 M NaZPZ07 with the DNA-negative 0.1 ml, and 0.5 ml of cold 10% TCA. The S27, but is only slightly affected after inmixture was incubated 30 min at 0’ and fection with the phage mutant bda 37
670
LAVI
AND MARCUS
DKA synthesis (Roscoe, 196913; Marcus and N&on, 1971). Addition of the drug 13 min after infection does not affect the arrest of host DNA synthesis. As shown in Table 2, arrest of host DNA synthesis in cells infected with S27, as determined in toluenized cells, can be prevented if chioramphenicol (50 rg/ml) is added at the time of infection. Addition of the drug 15 min after infection does not affect the inhibition of host, DNA synthesis. DXA
FIG. 1. Rate of DNA synthesis in toluenized preparations of uninfected and phage-infected Pal- cells. Cells were grown at 37”, infected, treated with toluene and assayed as described in Materials and Methods. Uninfected Bacillus subtilis 168 pol I- (0). Cells infected with phage mutant S27 (a). Cells infected with phage mut,ant bda 37 (A). derived from S27. These results wcrc obtained in experiments carried out in minimal medium with B. subtilis 3610 (Marcus
and Newlon, 1971). Similar results were obtained in rich medium with B. subtilis 168 Pol I- strain (data not presented). The results presented in Fig. 1 show that DNA synthesis in a toluenized preparation of S27 infected cells is inhibited several minutes aft’er infection. DNA synthesis continues in a toluenized preparation of bda 37 infected cells albeit at a lower rate as compared with the rate of synthesis in uninfected cells. These results arc similar to those obtained in vivo (Marcus and Newlon, 1971). CsCl gradients of the DNA isolated from toluenc-treated cells infected with phage S27 or bda 37 showed that the DNA synthesized in the toluenized infected cells had the buoyant density of the bacterial DNA (p = 1.70 g/cm3). Effect cf Chloramphenicol Treatment on the Arrest of Host DLVA Synthesis in Cells Infected with Phage Xd7 Chloramphenicol added at the time of infection prevents the shutoff of bacterial
Synthesis in Cells Infected with the Wild-Type Phage +e or with the dTTPaseless Phage Mutant 5/18/R
The wild-type phage +e induces the synthesis of dTTPase a few minutes after infection (Roscoe, 1969a; b). Thymine incorporation into DNA in vivo stops 6 min after infection of cells wit,h the wild-type phage at 37” (Roscoe, 196913). As mentioned earlier, synthesis of host DNA in cells infected with the dTTPaseless phage S27 was arrested only 14 min after infection. The difference in the time of arrest of host DNA synt,hesis between cells infected with 4e and cells infected with S27 was attributed to the depletion of the dTTP pool by dTTPase in 4e-infected cells. In accordance with this result, it was found that crude extracts of $e-infected cells TABLE
2
EFFECT OF CHLOR.~MPHENICOL (50 pg/ml) TREBTMENT ON THE INCORPORATION OF aH-dTMP INTO DNA IN TOLUENIZED PREPARATIONS OF UNINFECTED AND S27-INFECTED CELLS'
Treatment
No chloramphenicol added Chloramphenicol added at time of in fection Chloramphenicol added 15 min after infection
Uninfected cells 3H-dTMP PG$,s,/
S27 infected cells 3H-dTMP p,mp;/
50
10
33
35
55
15
y Cells were grown at 37”, infected, treated with toluene 20 min after infection and assayed as described in Materials and Methods.
ARREST
OF HOST
did not exhibit any DNA polymerase activity when the labeled nucleotide was dTTP (Marcus, unpublished results). The course of DNA synthesis in cells infected with 4e and with the dTTPaseless DNA-positive phage mutant 5/18/R was determined in toluene-treated cells. The results presented in Fig. 2 show that in cells infected with 4e, incorporation of labeled dTMP into DNA is inhibited very early after infection. On the other hand. in cells infected with
30 FIG. 2. Rate of DNA synthesis in toluenieed preparations of uninfected and phage infected Pal- cells. Cells were grown at 37”, infected, treated with toluene and assayed as described in Materials and Methods. Uninfected BaciZZus subtilis 168 pal I- (0). Cells infected with wildtype phage +e (0). Cells infected with phage mutant 5/18/R (A).
RATE OF DNA
30
671
SYNTHESIS
5/18/R the rate of incorporation of 3HdTMP remains considerably high. The difference between the rate of 3H-dTMP incorporation into DNA in cells infected with the dTTPaseless DNA-negative mutant S27 and in cells infected with the dTTPaseless DNA-positive mutant 5/18/R is to be expected if what is synthesized in 5/18/R-infected cells is phage DNA with thymine being incorporated into DNA instead of hvdroxvmethvluracil. This possibility” was investigated furt,her. A DNA thermosensitive phage mutant al was isolated from 5/18/R. Both 5/18/R and the double mutant phage al arrested host DNA synthesis in viva at 30” 20 min after infection and at 45” 14 min after infection. At 30” al grew almost normally, but at 45” no phage DNA was made (data not presented). The data shown in Table 3 indicate that the rate of 3H-dTMP incorporation into DNA in a toluenized preparation of cells infected with 5/18/R is 68% of the rate in uninfected cells at 30” and 51.6% at 45’. In al infected cells the rate of 3H-dTMP incorporation into DNA at 30” is 38% of the rate in uninfected cells, while at 45” the rate is only 14.6%. These results indicate that phage DNA containing thymine is synthesized in a toluenized preparation of cells infected with 5/18/R. The reduction in the rates of DNA synthesis at 45“ in comparison to the rate at 30”, in both uninfected and 5/18/R infected cells, reflects the general sensitivity
TABLE 3 SYNTHESIS IN TOLUENIZED PREPARATIONS OF UNINFECTED CELLS AND CELLS INFECTED WITH 5/18/R OR al PHAGE MUTANTS~
Time afteti Temperature of Uninfected cells infection (min) growth after 3H-dTMP infection (“C) pmoles per 66 min
22
DNA
30 45
44.5 24.6
Cells infected with 5/18/R
Cells infected with al
3H-dTMP pmoles per 66 min
unEfe?ted
WdTMP pmoles per 66 min
un?fe$t ed
30.3 12.7
68 51.6
17.1 3.6
38.4 14.6
Q Cells were grown at 30”, infected either at 30” or at 45”, treated with toluene at room temperature and assayed as described in Materials and Methods at the temperature of growth after infection. b The samples were tolueniaed after full arrest of host DNA was established and before cell lysis set in.
672
LAVI TABLE
AND
4
EFFECT OF CENTRIFUGATION AND Wasmxvc: on DIALYSIS ON THE RATE OF DNA SYNTHESIS IN TOLUENIZEU PREPARATIONS OF UNINFECTW AND
PHAGE
Treatment
S27 INFECTED
CELLS~
Uninfected 3H-dTMP pmole/66 min
pmole/fKl min
43.0 45.5 44.0
7.4 7.2 7.3
None Centrifugation Dialysis
:27-7;;t;d
0 Cells were grown at 37” infected, and treated with toluene 25 min after infection. Aliquots were then treated in one of the following ways: (1) assayed immediately as described in Materials and Methods; (2) centrifuged and washed three times and assayed as above; (3) dialyzed and assayed as above.
of DNA synthesis high temperatures.
in toluenized
Mechanisms of Arrest of Host DNA
cells at Synthesis
The possibility that HlMdUMP or its derivatives interfere with host DNA synthesis was investigated by Marcus and Newlon (1971) using phage 527, which hardly makes any detectable amounts of the known phage enzymes including the deoxyuridylate hydroxymethylase (HM). The finding was that host DNA synthesis was still arrested in S27-infected cells, the kinetics of arrest being similar to that found in cells infected with dTTPaseless phage mutants. These results did not rule out the possibility that small amounts of HMdUMP were made even in S27-infected cells and interfered with host DNA synthesis. Our system of toluenized cells enabled us to investigate further this possibility. We determined the inhibition of host DNA synthesis in a toluenized preparation of S27 infected cells after either centrifugation and washing or dialysis. A toluenized preparation of uninfected cells served as a control. The results presented in Table 4 show that neither treatment could alleviate the inhibit,ion of host DNA synthesis in the infected cells. Another possibility tested was that the phage induces after infection the synthesis of a specific nucleasc that produces single-
MARCUS
strand breaks in the host chromosome. Thesck breaks are not repaired either because repair DNA synthesis by the DNA polymerase repair enzyme is inhibited in the infected cells or because the rate of repair DNA synthesis does not balance the rate of single-strand breaks production. The results presented in Fig. 3 show that the ATP-dependent. DNA synthesis in a toluenized preparation of 527 infected 168 cells is inhibited. The kinetics of inhibition are similar to those observed in Pal- infected cells (Fig. 1). The data presented in Table 5 show that the inhibition of DNA synthesis in a toluenized preparation of S27-infected 168 cells is not accompanied by induction of the ATP-independent repair DNA synthesis. Nevertheless, ATPindependent repair DNA synthesis can be induced by pancreatic DNase treatment in both toluenized preparations of uninfected or S27 infected cells. This induction is accompanied by reduction in the rate of ATP-dependent DNA synthesis. Preliminary experiments indicate that an unidentified factor that leaks out from toluenized preparations of S27 infected cells inhibits DNA synthesis when added
I 0
/
5
I I I 10 15 20 25 "INUTES AFTER lNFEC,lON
I 30
P-FIG. 3. Rate of DNA synthesis in toluenized preparations of uninfected and phage S27-infected 168 cells. Cells were grown at 37”, infected, treated with toluene and assayed as described in Materials and Methods. Uninfected Bacillus subtilis 168 Pol If (e). Cells infected with phage mutant 527 (0).
ARREST
OF HOST DNA SYNTHESIS TABLE
673
5
EFFECT OF PANCREATIC DEOXYRIBONUCLEASE ON THE RATE OF ATP DEPENDENT AND ATP INDEPENDENT DNA SYNTHESIS IN TOLUENIZED PREPARATIONS OF UNINFECTED AND S27 INFECTED CELLS” Uninfected
System
Complete - ATP +DNase -ATP $ DNase
3H-dTMP pmoles per 60 min
S27 infected aH-dTMP pmoles per 60 min
Total
Net ATP dependent
Total
55.2 14.4 109.3 86.8
40.8
16.7 7.0 77.4 71.0
22.5
Net ATP dependent 9.7 6.4
(1168 cells were grown at 37’, infected, treated with toluene 20 min after infection and assayed as described in Materials and Methods. Pancreatic DNase was present at 0.15 rg/0.15 ml reaction mixture where indicated. ATP was omitted where indicated. t,o toluenized uninfected cells. This factor does not leak out from toluenized preparations of bda 37 infected cells or 527 infected cells in which host DNA arrest was prevented by chloramphenicol. DISCUSSION
Host DNA Arrest Previous experiments showed that phage +e induces the synthesis of a protein which is involved in host DNA arrest (Marcus and Newlon, 1971). The nature of this protein and the mechanism of host DNA arrest is not known. Our results show that the inhibition of host DNA synthesis in cells infected with phage 527 (DNA negative; dTTPase-; HM-) persist in a toluenized preparation of the infected cells which are made accessible to the different nucleotide triphosphates. This inhibition can be prevented by the addition of chloramphenicol to the phageinfected cells at the time of infection. A toluenized preparation of bda 37 infected cells exhibit almost normal rate of host DNA synthesis. These results, which reflect faithfully the in vivo results, support, the conclusion reached by Marcus and Newlon (1971) that the mechanism of host DNA arrest is determined by a specific phage directed DNA arrest or bda protein (bacterial protein) apart from the depletion of dTTP by the phage-induced dTTPase. We have excluded the possibility that
the small amount of H1ldU1IP or its derivatives which were made in S27 infected cells caused the arrest of host DNA synthesis, since inhibition of host DNA synthesis persisted even after either centrifugation and washing or dialysis of the toluenized preparations of the infected cells. Arrest of host DNA synthesis in 4e infected cells was found not to be accompanied by the production of double-stranded breaks in the host, DNA (Roscoe, 1969b). Our results show that arrest of host DNA synthesis which was observed in a toluenized preparation of 527 infected cells was not accompanied by induction of repair DNA synthesis. Nevertheless, repair DNA synthesis, which depends on the DNA polymerase repair enzyme, could be induced by pancreatic DNase treatment (Moses and Richardson, 1970) in the toluenized preparation of S27-infected 168 cells. These resuIts indicate that host DNA arrest, in S27-infected cells is not caused by the production of single-strand breaks in the host DNA. We cannot exclude the possibility that the arrest of host, DNA synthesis depends on the production of specific single-strand breaks that are not a substrate for the DNA polymerase repair enzyme and therefore do not induce the ATP independent repair DNA synthesis. The factor that appears to leak out from toluenized preparations of S274nfected cells and inhibits DNA synthesis in toluenized uninfected cells may be the phage-
LAVI
674
AND MARCUS
directed bda protein. Purification and characterization of this factor may enable us to find out whether the mechanism of host DNA arrest determined by the phage bda protein depends on modification or inhibition of the bacterial replicase. Phage DNA Synthesis Previous in vivo experiments (Roscoe, 1969b; Marcus and Newlon, 1971; Marcus, unpublished results) showed that host DNA synthesis was arrested in cells infected with dTTPaseless DNA-positive phage mutants. Thymine that continued to be incorporated into DNA throughout the lytic cycle was found in phage DNA. Our results with toluenized preparations of strain 5/18/R (DNA positive dTTPaseless) infected cells showed that DNA synthesis continued throughout the experiments at a rate significantly higher than the residual rate of DNA synthesis observed in a toluenized preparation of S27 (DNA negative dTTPaseless) infected cells. Further investigation with the double mutant phage al (DNAts; dTTPaseless) showed that in a toluenized preparation of al infected cells DNA synthesis was arrested only at the restrictive temperature of phage DNA synthesis. These results indicate that in a toluenized preparation of 5/18/R infected cells host DNA synthesis is arrested and phage DNA synthesis occurs, with thymine being incorporated into phage DNA instead of hydroxymethyluracil . ACKNOWLEDGMENTS The authors wish to thank Dr. J. Gross for the gift of B. subtilis pol I- strain; Dr. S. E. Luria for many helpful suggestions; Drs. Y. S. Halpern and A. Ronen for helpful discussions; and Miss A. Nattenberg for her excellent technical assistance. This work was supported by a grant to Dr. M. Marcus from the Authority for Research and Development, Hebrew University (050322).
H.EFERENCES BILLEN, D., CARREIRA, L. B., and SILVERSTEIN, S.
(1971). Utilization of deoxyribonucleoside triphosphates in the cellular synthesis of DNA by Bacillus subtilis. Biochem. Biophys. Res. Commm. 43,1150-1157. BURGER, R. M. (1971). Toluene-treated Escherichia coli replicate only that DNA which wm about to be replicated in tivo. Proc. Nat. Acad. Sci. U.S.A. 68, 2124-2126. DE LUCIA, P., and CAIRNS, J. (1969). Isolation of an Escherichia coli strain with a mutation affecting DNA Polymerase. Nature (London) 224,
116&1166. GANESAN, A. T. (1968). Studies on the in vitro synthesis of transforming DNA. Proc. Nat. Acad. Sci. U.S.A. 61,1058-1065. KOHIYAMA, M., and KOLBER, A. R. (1970). Tem-
perature sensitive mutant of the DNA replication system in Escherichia coli. Nature (London) 228,1157-1160. MARCUS, M., and NEWLON, M. C. (1971). Control of DNA synthesis in Bacillus subtilis by phage +e. Virology 44,83-93. MATSUSHITA, T., WHITE, K. P., and SUEOKA, N. (1971). Chromosome replication in toluenized Bacillus subtilis cells. Nature (London) New Biol. 232, 111-114. MORDOH, J., HIROTA, Y., and JACOB, F. (1970). On the process of cellular division in Escherichia coli. V. Incorporation of deoxynucleoside triphosphates by DNA thermosensitive mutants of Escherichia coli also lacking DNA polymerase activity. Proc. Nat. Acad. Sci. U.S.A. 67, 773778. MOSES, R. E., and RICHARDSON, C. C. (1970). Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc. Nat. Acad. Sci. U.S.A. 67,674-681. ROSCOE, D. H. (1969a). Thymidine triphosphate
nucleotidohydrolase: a phage-induced enzyme in Bacillus subtilis. Virology 38, 520-526. ROSCOE, D. H. (1969b). Synthesis of DNA in phage-infected Bacillus subtilis. Virology 38, 527-537. ROSCOE, D. H., and TUCKER, R. G. (1966). The biosynthesis of 5-hydroxymethyluridylic acid in bacteriophage-infected Bacillus subtilis. Virology29,157-166.
49,
668-674 (1972)
Arrest
of Host
DNA
Synthesis with
URl Department
LAVI
AND
of Genetics,
in Bacillus
Phage
Infected
4e
MENASHE
The Hebrew
sobtilis
MARCUS
University,
Jerusalem,
Israel
Accepted May 26, 19% Arrest of host DNA synthesis in Bacillus subtilis infected with the virulent phage be is dependent on the synthesis of a phage-coded protein (Marcus and Newlon, 1971). The mechanism of host DNA arrest was studied in toluene-treated B. subtilis cells. The results showed that preparations of toluenized phage infected cells manifest faithfully the kinetics and specificity of host DNA arrest and the appearance of phage DNA synthesis. The arrest of host DNA synthesis is not caused by the production in the host DNA of single strand breaks that can be repaired by the DNA polymerase repair enzyme (Pol I). Neither hydroxymethyl deoxyuridylate (HMdUMP) nor probably any other small molecule is responsible for host DNA arrest. INTRODUCTION
Bacteriophage t$e, which infects Bacillus subtilis, arrests host DNA synthesis a few minutes after infection (Roscoe, 196913; Marcus and Newlon, 1971). This virulent phage contains hydroxymethyluracil (HMU) instead of thymine in its DNA, and induces the synthesis of several proteins, including an inhibitor of the bacterial thymidylate synthetase and a deoxythymidine nucleotidohydrolase (dTTPase) early after infection (Roscoe and Tucker, 1966; Roscoe, 1969a). The arrest of host DNA synthesis in the infected cells was shown not to be dependent on the depletion of the dTTP pool in the infected cells due to the action of thymidylate synthetase inhibitor or dTTPase. Inhibition occurred in thymine requiring bacterial mutants infected with dTTPaseless phage mutants in the presence of exogenous thymine (Roscoe, 1969b; Marcus and Newlon, 1971). Depletion of the dTTP pool in the infected cells is probably important for the normal growth cycle of the phage. Phage mutants that do not destroy the dTTP pool in the infected cells incorporate thymine into DNA (Roscoe, 1969b; Marcus and Newlon, 1971). Replacement of HMU by thymine 668 Copyright @ ltl72 by Academic Press, Inc. All rights of reproduction in any form reserved.
above a critical level interferes with phage DNA synthesis (Marcus, unpublished resuits) . Further investigation on the mechanism of host DNA arrest was based on studies of DNA synthesis in toluene treated cells of B. subtilis wild-type strain and its DNA polymerase negative mutant (Pol I-). The results discussed in this paper show that: (1) The kinetics of host and phage DNA synthesis in toluene treated cells are similar to those obtained in vivo. (2) The absence of hydroxymethyl deoxyuridylate (HMdUMP) or its derivatives in cells infect’ed with phage does not prevent host DNA arrest. (3) Host DNA arrest is not caused by the production of repairable single strand breaks in the host chromosome. MATERIALS
AND
METHODS
Microorganisms. The bacterial strains are derivatives of B. subtilis 168. The phages are derivatives of 4e. A list of the strains is presented in Table 1. Media. LB Broth (Marcus and Newlon, 1971) was used throughout this work. Assay of DlVA synthesis in toluene treated uninfected and, infected cells. Bacteria were grown in LB broth to Fi X 107 cells/ml.
TABLE
PHAGE AND
Bacteria 168 168 Phage +e S27 bda37 (derived from 527) 5/18/R al (derived from
OF HOST
STRAINS
USED~
1
BACTERIAL
Strain
ARREST
Relevant genotype
Wild type PO1 IWild type sus DNA-negative dTTPase-n HMsus DNA-newtive dTTPaseHMdTTPasedTTPaseDNAtS
bda-
5/18/R) a Abbreviations: Pol I, DNA polymerase (repair enzyme) ; dTTPase, deoxythymidine triphosphate nucleotidohydrolase; HM, deoxyuridylate hydroxymethylase; bda, bacterial DNA arrest.
DNA
SYNTHESIS
669
filtered on Whatman GF/C filters, washed with 15 ml of ice-cold 1% TCA, dried and counted in a 3380 Packard Liquid Scintillation Spectrometer. Density gradient centrifugation. CsCl equilibrium gradients were performed as described by Roscoe (1969b). CsCl (99.9%) was purchased from Varlacoid Chemical Company. Centrifugation was carried out in a L2-50 Beckman Preparative Ultracentrifuge with a SW 50.1 rotor for 48 hr at 35,000 rpm at 22”. RESULTS
In a previous paper (Marcus and Newlon, 1971) the possibility that the arrest of host DNA synthesis by phage $e resulted from inhibition of the host replication complex by the phage was explored. Activity of DNA polymerase was determined in crude and partially purified extracts prepared according to Ganesan (1968). Specific activities of DNA polymerase in extracts of uninfected cells and cells infected with the DNA-negative phage mutant S27 were similarly high and probably represented the activity of a repair enzyme (Marcus and Newlon, 1971; De Lucia and Cairns, 1969). It has since been found that DNA replication can be studied in toluenetreated cells of E. coli (Moses and Richardson, 1970; Mordoh et al., 1970; Kohiyama and Kolber, 1970; Burger, 1971) and B. subt&s (Matsushita et al., 1970; Billen et al., 1971). We decided to use a pal I- mutant of B. subtilis in order to eliminate the possibility of repair polymerase activity which occurs in toluene-treated cells under certain conditions (Moses and Richardson, 1970). Dr. J. Gross kindly supplied us with the B. subtilis pal Imutant. DNA synthesis in this mutant after toluene treatment was found to be dependent on the presence of ATP and was inhibited by p-chloromercuribenzoate.
Cell density was determined by direct counting in a Petroff-Hauser cell. Phage infection was carried out at this cell density at a multiplicity of infection (m.0.i) of 10. In all experiments, survival of cells 10 min after infection was l-5%. At various times, 15-ml aliquots were washed and suspended in 1 ml of phosphate buffer 0.05 M pH 7.4. Toluene (10 ~1) was added and the suspension was vigorously agitated on a Vortex mixer for 1 min at room temperature. The reaction mixture was based on that of Kohiyama and Kolber (1970) with some modifications: 0.05 M Tris buffer pH 7.4; 0.005 M MgC12; 0.09 M KCI; 0.001 M dithiothreitol; 1 X lop3 M ATP; 3 X lo+ M of: dATP, dCTP, dGTP; 7 X 10-e M dTTP; 0.15 #Zi 3H-dTTP; 0.001 M NaN3; 3.5-7 X lo7 cells. The cells were added to the mixture immediately after toluene treatment. Water was added to a final volume of 0.15 ml. The reaction was terminated after 30 min at the specified temperature by addition of Host DNA Synthesis in Cells Infected with 0.15 ml of 1 M NaOH and 0.02 M ethylenePhage Mutants 827 and bdaS7 diaminetetraacetic acid. The mixture was Host DNA synthesis in vivo at 37” is heated for 30 set at loo”, cooled immearrested at about 14 min after infection diately to 0” and the following additions phage mutant were made: 1 M HCI 0.15 ml, 0.1 M NaZPZ07 with the DNA-negative 0.1 ml, and 0.5 ml of cold 10% TCA. The S27, but is only slightly affected after inmixture was incubated 30 min at 0’ and fection with the phage mutant bda 37
670
LAVI
AND MARCUS
DKA synthesis (Roscoe, 196913; Marcus and N&on, 1971). Addition of the drug 13 min after infection does not affect the arrest of host DNA synthesis. As shown in Table 2, arrest of host DNA synthesis in cells infected with S27, as determined in toluenized cells, can be prevented if chioramphenicol (50 rg/ml) is added at the time of infection. Addition of the drug 15 min after infection does not affect the inhibition of host, DNA synthesis. DXA
FIG. 1. Rate of DNA synthesis in toluenized preparations of uninfected and phage-infected Pal- cells. Cells were grown at 37”, infected, treated with toluene and assayed as described in Materials and Methods. Uninfected Bacillus subtilis 168 pol I- (0). Cells infected with phage mutant S27 (a). Cells infected with phage mut,ant bda 37 (A). derived from S27. These results wcrc obtained in experiments carried out in minimal medium with B. subtilis 3610 (Marcus
and Newlon, 1971). Similar results were obtained in rich medium with B. subtilis 168 Pol I- strain (data not presented). The results presented in Fig. 1 show that DNA synthesis in a toluenized preparation of S27 infected cells is inhibited several minutes aft’er infection. DNA synthesis continues in a toluenized preparation of bda 37 infected cells albeit at a lower rate as compared with the rate of synthesis in uninfected cells. These results arc similar to those obtained in vivo (Marcus and Newlon, 1971). CsCl gradients of the DNA isolated from toluenc-treated cells infected with phage S27 or bda 37 showed that the DNA synthesized in the toluenized infected cells had the buoyant density of the bacterial DNA (p = 1.70 g/cm3). Effect cf Chloramphenicol Treatment on the Arrest of Host DLVA Synthesis in Cells Infected with Phage Xd7 Chloramphenicol added at the time of infection prevents the shutoff of bacterial
Synthesis in Cells Infected with the Wild-Type Phage +e or with the dTTPaseless Phage Mutant 5/18/R
The wild-type phage +e induces the synthesis of dTTPase a few minutes after infection (Roscoe, 1969a; b). Thymine incorporation into DNA in vivo stops 6 min after infection of cells wit,h the wild-type phage at 37” (Roscoe, 196913). As mentioned earlier, synthesis of host DNA in cells infected with the dTTPaseless phage S27 was arrested only 14 min after infection. The difference in the time of arrest of host DNA synt,hesis between cells infected with 4e and cells infected with S27 was attributed to the depletion of the dTTP pool by dTTPase in 4e-infected cells. In accordance with this result, it was found that crude extracts of $e-infected cells TABLE
2
EFFECT OF CHLOR.~MPHENICOL (50 pg/ml) TREBTMENT ON THE INCORPORATION OF aH-dTMP INTO DNA IN TOLUENIZED PREPARATIONS OF UNINFECTED AND S27-INFECTED CELLS'
Treatment
No chloramphenicol added Chloramphenicol added at time of in fection Chloramphenicol added 15 min after infection
Uninfected cells 3H-dTMP PG$,s,/
S27 infected cells 3H-dTMP p,mp;/
50
10
33
35
55
15
y Cells were grown at 37”, infected, treated with toluene 20 min after infection and assayed as described in Materials and Methods.
ARREST
OF HOST
did not exhibit any DNA polymerase activity when the labeled nucleotide was dTTP (Marcus, unpublished results). The course of DNA synthesis in cells infected with 4e and with the dTTPaseless DNA-positive phage mutant 5/18/R was determined in toluene-treated cells. The results presented in Fig. 2 show that in cells infected with 4e, incorporation of labeled dTMP into DNA is inhibited very early after infection. On the other hand. in cells infected with
30 FIG. 2. Rate of DNA synthesis in toluenieed preparations of uninfected and phage infected Pal- cells. Cells were grown at 37”, infected, treated with toluene and assayed as described in Materials and Methods. Uninfected BaciZZus subtilis 168 pal I- (0). Cells infected with wildtype phage +e (0). Cells infected with phage mutant 5/18/R (A).
RATE OF DNA
30
671
SYNTHESIS
5/18/R the rate of incorporation of 3HdTMP remains considerably high. The difference between the rate of 3H-dTMP incorporation into DNA in cells infected with the dTTPaseless DNA-negative mutant S27 and in cells infected with the dTTPaseless DNA-positive mutant 5/18/R is to be expected if what is synthesized in 5/18/R-infected cells is phage DNA with thymine being incorporated into DNA instead of hvdroxvmethvluracil. This possibility” was investigated furt,her. A DNA thermosensitive phage mutant al was isolated from 5/18/R. Both 5/18/R and the double mutant phage al arrested host DNA synthesis in viva at 30” 20 min after infection and at 45” 14 min after infection. At 30” al grew almost normally, but at 45” no phage DNA was made (data not presented). The data shown in Table 3 indicate that the rate of 3H-dTMP incorporation into DNA in a toluenized preparation of cells infected with 5/18/R is 68% of the rate in uninfected cells at 30” and 51.6% at 45’. In al infected cells the rate of 3H-dTMP incorporation into DNA at 30” is 38% of the rate in uninfected cells, while at 45” the rate is only 14.6%. These results indicate that phage DNA containing thymine is synthesized in a toluenized preparation of cells infected with 5/18/R. The reduction in the rates of DNA synthesis at 45“ in comparison to the rate at 30”, in both uninfected and 5/18/R infected cells, reflects the general sensitivity
TABLE 3 SYNTHESIS IN TOLUENIZED PREPARATIONS OF UNINFECTED CELLS AND CELLS INFECTED WITH 5/18/R OR al PHAGE MUTANTS~
Time afteti Temperature of Uninfected cells infection (min) growth after 3H-dTMP infection (“C) pmoles per 66 min
22
DNA
30 45
44.5 24.6
Cells infected with 5/18/R
Cells infected with al
3H-dTMP pmoles per 66 min
unEfe?ted
WdTMP pmoles per 66 min
un?fe$t ed
30.3 12.7
68 51.6
17.1 3.6
38.4 14.6
Q Cells were grown at 30”, infected either at 30” or at 45”, treated with toluene at room temperature and assayed as described in Materials and Methods at the temperature of growth after infection. b The samples were tolueniaed after full arrest of host DNA was established and before cell lysis set in.
672
LAVI TABLE
AND
4
EFFECT OF CENTRIFUGATION AND Wasmxvc: on DIALYSIS ON THE RATE OF DNA SYNTHESIS IN TOLUENIZEU PREPARATIONS OF UNINFECTW AND
PHAGE
Treatment
S27 INFECTED
CELLS~
Uninfected 3H-dTMP pmole/66 min
pmole/fKl min
43.0 45.5 44.0
7.4 7.2 7.3
None Centrifugation Dialysis
:27-7;;t;d
0 Cells were grown at 37” infected, and treated with toluene 25 min after infection. Aliquots were then treated in one of the following ways: (1) assayed immediately as described in Materials and Methods; (2) centrifuged and washed three times and assayed as above; (3) dialyzed and assayed as above.
of DNA synthesis high temperatures.
in toluenized
Mechanisms of Arrest of Host DNA
cells at Synthesis
The possibility that HlMdUMP or its derivatives interfere with host DNA synthesis was investigated by Marcus and Newlon (1971) using phage 527, which hardly makes any detectable amounts of the known phage enzymes including the deoxyuridylate hydroxymethylase (HM). The finding was that host DNA synthesis was still arrested in S27-infected cells, the kinetics of arrest being similar to that found in cells infected with dTTPaseless phage mutants. These results did not rule out the possibility that small amounts of HMdUMP were made even in S27-infected cells and interfered with host DNA synthesis. Our system of toluenized cells enabled us to investigate further this possibility. We determined the inhibition of host DNA synthesis in a toluenized preparation of S27 infected cells after either centrifugation and washing or dialysis. A toluenized preparation of uninfected cells served as a control. The results presented in Table 4 show that neither treatment could alleviate the inhibit,ion of host DNA synthesis in the infected cells. Another possibility tested was that the phage induces after infection the synthesis of a specific nucleasc that produces single-
MARCUS
strand breaks in the host chromosome. Thesck breaks are not repaired either because repair DNA synthesis by the DNA polymerase repair enzyme is inhibited in the infected cells or because the rate of repair DNA synthesis does not balance the rate of single-strand breaks production. The results presented in Fig. 3 show that the ATP-dependent. DNA synthesis in a toluenized preparation of 527 infected 168 cells is inhibited. The kinetics of inhibition are similar to those observed in Pal- infected cells (Fig. 1). The data presented in Table 5 show that the inhibition of DNA synthesis in a toluenized preparation of S27-infected 168 cells is not accompanied by induction of the ATP-independent repair DNA synthesis. Nevertheless, ATPindependent repair DNA synthesis can be induced by pancreatic DNase treatment in both toluenized preparations of uninfected or S27 infected cells. This induction is accompanied by reduction in the rate of ATP-dependent DNA synthesis. Preliminary experiments indicate that an unidentified factor that leaks out from toluenized preparations of S27 infected cells inhibits DNA synthesis when added
I 0
/
5
I I I 10 15 20 25 "INUTES AFTER lNFEC,lON
I 30
P-FIG. 3. Rate of DNA synthesis in toluenized preparations of uninfected and phage S27-infected 168 cells. Cells were grown at 37”, infected, treated with toluene and assayed as described in Materials and Methods. Uninfected Bacillus subtilis 168 Pol If (e). Cells infected with phage mutant 527 (0).
ARREST
OF HOST DNA SYNTHESIS TABLE
673
5
EFFECT OF PANCREATIC DEOXYRIBONUCLEASE ON THE RATE OF ATP DEPENDENT AND ATP INDEPENDENT DNA SYNTHESIS IN TOLUENIZED PREPARATIONS OF UNINFECTED AND S27 INFECTED CELLS” Uninfected
System
Complete - ATP +DNase -ATP $ DNase
3H-dTMP pmoles per 60 min
S27 infected aH-dTMP pmoles per 60 min
Total
Net ATP dependent
Total
55.2 14.4 109.3 86.8
40.8
16.7 7.0 77.4 71.0
22.5
Net ATP dependent 9.7 6.4
(1168 cells were grown at 37’, infected, treated with toluene 20 min after infection and assayed as described in Materials and Methods. Pancreatic DNase was present at 0.15 rg/0.15 ml reaction mixture where indicated. ATP was omitted where indicated. t,o toluenized uninfected cells. This factor does not leak out from toluenized preparations of bda 37 infected cells or 527 infected cells in which host DNA arrest was prevented by chloramphenicol. DISCUSSION
Host DNA Arrest Previous experiments showed that phage +e induces the synthesis of a protein which is involved in host DNA arrest (Marcus and Newlon, 1971). The nature of this protein and the mechanism of host DNA arrest is not known. Our results show that the inhibition of host DNA synthesis in cells infected with phage 527 (DNA negative; dTTPase-; HM-) persist in a toluenized preparation of the infected cells which are made accessible to the different nucleotide triphosphates. This inhibition can be prevented by the addition of chloramphenicol to the phageinfected cells at the time of infection. A toluenized preparation of bda 37 infected cells exhibit almost normal rate of host DNA synthesis. These results, which reflect faithfully the in vivo results, support, the conclusion reached by Marcus and Newlon (1971) that the mechanism of host DNA arrest is determined by a specific phage directed DNA arrest or bda protein (bacterial protein) apart from the depletion of dTTP by the phage-induced dTTPase. We have excluded the possibility that
the small amount of H1ldU1IP or its derivatives which were made in S27 infected cells caused the arrest of host DNA synthesis, since inhibition of host DNA synthesis persisted even after either centrifugation and washing or dialysis of the toluenized preparations of the infected cells. Arrest of host DNA synthesis in 4e infected cells was found not to be accompanied by the production of double-stranded breaks in the host, DNA (Roscoe, 1969b). Our results show that arrest of host DNA synthesis which was observed in a toluenized preparation of 527 infected cells was not accompanied by induction of repair DNA synthesis. Nevertheless, repair DNA synthesis, which depends on the DNA polymerase repair enzyme, could be induced by pancreatic DNase treatment (Moses and Richardson, 1970) in the toluenized preparation of S27-infected 168 cells. These resuIts indicate that host DNA arrest, in S27-infected cells is not caused by the production of single-strand breaks in the host DNA. We cannot exclude the possibility that the arrest of host, DNA synthesis depends on the production of specific single-strand breaks that are not a substrate for the DNA polymerase repair enzyme and therefore do not induce the ATP independent repair DNA synthesis. The factor that appears to leak out from toluenized preparations of S274nfected cells and inhibits DNA synthesis in toluenized uninfected cells may be the phage-
LAVI
674
AND MARCUS
directed bda protein. Purification and characterization of this factor may enable us to find out whether the mechanism of host DNA arrest determined by the phage bda protein depends on modification or inhibition of the bacterial replicase. Phage DNA Synthesis Previous in vivo experiments (Roscoe, 1969b; Marcus and Newlon, 1971; Marcus, unpublished results) showed that host DNA synthesis was arrested in cells infected with dTTPaseless DNA-positive phage mutants. Thymine that continued to be incorporated into DNA throughout the lytic cycle was found in phage DNA. Our results with toluenized preparations of strain 5/18/R (DNA positive dTTPaseless) infected cells showed that DNA synthesis continued throughout the experiments at a rate significantly higher than the residual rate of DNA synthesis observed in a toluenized preparation of S27 (DNA negative dTTPaseless) infected cells. Further investigation with the double mutant phage al (DNAts; dTTPaseless) showed that in a toluenized preparation of al infected cells DNA synthesis was arrested only at the restrictive temperature of phage DNA synthesis. These results indicate that in a toluenized preparation of 5/18/R infected cells host DNA synthesis is arrested and phage DNA synthesis occurs, with thymine being incorporated into phage DNA instead of hydroxymethyluracil . ACKNOWLEDGMENTS The authors wish to thank Dr. J. Gross for the gift of B. subtilis pol I- strain; Dr. S. E. Luria for many helpful suggestions; Drs. Y. S. Halpern and A. Ronen for helpful discussions; and Miss A. Nattenberg for her excellent technical assistance. This work was supported by a grant to Dr. M. Marcus from the Authority for Research and Development, Hebrew University (050322).
H.EFERENCES BILLEN, D., CARREIRA, L. B., and SILVERSTEIN, S.
(1971). Utilization of deoxyribonucleoside triphosphates in the cellular synthesis of DNA by Bacillus subtilis. Biochem. Biophys. Res. Commm. 43,1150-1157. BURGER, R. M. (1971). Toluene-treated Escherichia coli replicate only that DNA which wm about to be replicated in tivo. Proc. Nat. Acad. Sci. U.S.A. 68, 2124-2126. DE LUCIA, P., and CAIRNS, J. (1969). Isolation of an Escherichia coli strain with a mutation affecting DNA Polymerase. Nature (London) 224,
116&1166. GANESAN, A. T. (1968). Studies on the in vitro synthesis of transforming DNA. Proc. Nat. Acad. Sci. U.S.A. 61,1058-1065. KOHIYAMA, M., and KOLBER, A. R. (1970). Tem-
perature sensitive mutant of the DNA replication system in Escherichia coli. Nature (London) 228,1157-1160. MARCUS, M., and NEWLON, M. C. (1971). Control of DNA synthesis in Bacillus subtilis by phage +e. Virology 44,83-93. MATSUSHITA, T., WHITE, K. P., and SUEOKA, N. (1971). Chromosome replication in toluenized Bacillus subtilis cells. Nature (London) New Biol. 232, 111-114. MORDOH, J., HIROTA, Y., and JACOB, F. (1970). On the process of cellular division in Escherichia coli. V. Incorporation of deoxynucleoside triphosphates by DNA thermosensitive mutants of Escherichia coli also lacking DNA polymerase activity. Proc. Nat. Acad. Sci. U.S.A. 67, 773778. MOSES, R. E., and RICHARDSON, C. C. (1970). Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc. Nat. Acad. Sci. U.S.A. 67,674-681. ROSCOE, D. H. (1969a). Thymidine triphosphate
nucleotidohydrolase: a phage-induced enzyme in Bacillus subtilis. Virology 38, 520-526. ROSCOE, D. H. (1969b). Synthesis of DNA in phage-infected Bacillus subtilis. Virology 38, 527-537. ROSCOE, D. H., and TUCKER, R. G. (1966). The biosynthesis of 5-hydroxymethyluridylic acid in bacteriophage-infected Bacillus subtilis. Virology29,157-166.