- Email: [email protected]
Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative from in dna mutants of Escherichia coli
149
Biochimica et Biophysica Acta, 476 (1977) 149--155 © Elsevier/North-Holland Biomedical Press
BBA 98928 CONVERSION OF BACTERIOPHAGE G4 SINGLE-STRANDED VIRAL DNA TO DOUBLE-STRANDED REPLICATIVE FORM IN dna MUTANTS OF ESCHERICHIA COLI
KEN-ICHI K O D A I R A
and A K I R A
TAKETO
*
Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920 (Japan) (Received December 7th, 1976)
Summary Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escherichia coli. In dna ÷ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 #g/ml of rifampicin or 25 pg/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thevmosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43°C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.
Introduction Various enzymes and regulatory proteins necessary for host DNA synthesis are required for replication of single-stranded DNA phages as well. Thus, DNA polymerase III (dnaE gene product), the dnaG gene product, dnaZ protein, ribonucleotide reductase (nrdA gene product), and DNA ligase (lig gene product) are involved in replication of the St-1 group of phages including q~K [1], CXtB [2], and a3 (unpublished result). In addition to these functions, dnaB and dnaC(D) products are indispensable for replication of ¢X174 phage and other phages such as CA, G6, CB [1], S13 and ¢R (unpublished observation). Furthermore, all these phages require the rep function which is not required for replication of host bacteria. Replication of single-stranded (SS) DNA phages consists of three steps: conversion of single-stranded to parental replicative * To wh om requests for reprints s h o u l d b e sent.
150 form RF(I), subsequent replication of progeny RF(II) and asymmetrical synthesis of SS (III) [3]. In the case of ~X174, biochemical and genetic mechanisms operative in each step have been clarified considerably [4]. Recently, Zechel et al. [5] have reported that conversion of phage G4 DNA to RF II in vitro depends on three host proteins i.e dnaG protein, DNA binding protein and DNA polymerase III holoenzyme (which is known to be a complex of four discrete proteins). Regardless of the apparent simplicity of step I reaction in vitro, the host factor requirement in vivo is u n k n o w n for bacteriophage G4. The permissive growth temperature of phage G4 {phages G13, G14 and U3 as well) is relatively low and, at 42--43°C viral multiplication is severely restricted even in dna ÷ cells (unpublished observation). The enzymatic reaction involved in step I, however, occurs at the temperatures. In this report we have determined the host genetic factors required for synthesis of the parental replicative form in vivo. Materials and Methods Phage and bacterial strains. Bacteriophage G4 [6] was generously furnished by Dr. G.N. Godson. Escherichia coli H502 dna ÷ [7], C-2307 dnaA [8] and C2309 dnaG [8] were kindly provided by Dr. R. Calendar, strains LD312 dnaB [9], LD332 dnaC(D) [10] and LD301 dnaE [7] by Dr. L.B. Dumas, HF4704S dnaH [11] by Dr. T. Komano, C727 dnaZ [12] by Dr. J.R. Walker and strain D43 rep [13] by Dr. D.T. Denhardt. Preparation o f ~4C-labeled G4. Cells of E. coli H502 t hy- growing exponentially in TCG * medium [14] s lpplemented with 2 pg/ml of thymine were collected, washed, and resuspended in 50 ml of TCG medium at a density of A660 = 0.75. To the culture, 0.5 ml of 1 M CaC12, 1.5 • l 0 s plaque-forming units of phage G4, and 0.05 mCi of [~4C]thymine were added and incubation was continued at 37°C, with shaking. After lysis, EDTA was added to 20 mM and the lysate was centrifuged at 40 000 rev./min for 1 h. From the sediment, phages were extracted with 0.5% sodium dodecyl sulfate in 0.1 M NaCl/0.05 M Tris • HC1/0.01 M EDTA (pH 7.5), and subjected to several cycles of centrifugation at 14 000 rev./min and 40 000 rev./min. After treatment with 10 pg/ml of DNAase at 37°C for 1 h, the phages were layered on a 5 ml of 5--20% sucrose density gradient in dilution fluid [15] and centrifuged in a Hitachi RPS-50 rotor at 35 000 rev./min for 35 min. The gradient was collected dropwise from the b o t t o m and assayed for both ['4C] and plaque formation. The peak fractions were pooled, diluted with nutrient broth and stored at 4°C. The specific activity of the purified phage was 2 • 10 -6 cpm per plaque-forming unit. Infection. Bacteria were grown at 30°C in nutrient broth, with shaking, to a density of A6~o = 0.25 and divided into two 10 ml portions. One portion was shifted to 43°C and the other kept at 30°C. Each culture received 0.1 ml of 1 M CaC12 and, 10 min after the temperature shift, infected with ~4C-labeled phage G4 at a multiplicity of four. When initiation-defective dnaA or dnaH mutants of E. coli were infected, cells were pregrown at 43°C for 1 h. The infected culture was incubated for 15 min at 30°C or for 20 min at 43°C, with shaking. • TCG, Tris]casamino acids[~lucose.
151 Cell lysis and D N A extraction. Infection was terminated by pouring each culture into an equal volume of ice-cold 75% ethanol in 0.05 M Tris • HC1/0.02 M EDTA (pH 8.0). The cells were harvested and washed with chilled 0.05 M Tris • HC1/0.01 M EDTA (pH 8.0) by a brief centrifugation and suspended in the buffer. Lysozyme was added to a final concentration of 0.25 mg/ml and the mixture was kept at 0°C for 10 min and then at room temperature for 10 min. After addition of 0.5% sodium dodecyl sulfate, the mixture was incubated for 30 min at room temperature and treated further with 0.5 mg/ml of autodigested pronase for 4.5 h at 37°C. The sample was subsequently treated with an equal volume of 90% phenol to extract nucleic acids. After removal of residual phenol with ether, NaC1 was added to a final concentration of 1 M and the mixture was allowed to stand for 2 h at 0°C. The precipitated bacterial DNA was removed by a low speed centrifugation and the supernatant was subjected to sedimentation analysis. Analysis o f D N A on sucrose gradients. The DNA sample (0.1 ml) was layered onto a 5 ml of 5--20% sucrose density gradient in 0.05 M Tris • HCI/0.002 M EDTA/1 M NaC1 (pH 7.2) and centrifuged in a Hitachi RPS-50 rotor at 40 000 rev./min for 4 h, at 4°C. Fractions were collected from the b o t t o m of the gradient. D e t e r m i n a t i o n o f radioactivity. Aliquots from sucrose gradient fractions were spotted on glass filter paper, dried, and counted in a Nuclear Chicago liquid scintillation spectrometer. The scintillation fluid contained, per liter of toluene, 4 g of 2,5-diphenyloxazole and 0.1 g of 1.4-bis(2-(5-phenyloxazolyl))benzene. Others. [~4C]Thymine (58 Ci/mol) was purchased from the Radiochemical Centre. Rifampicin was obtained from Daiichi Pharmaceutical Co. Tokyo. Phage G4 single-stranded DNA was extracted from the purified phage by phenol method. The sources of other materials were as previously described [16,17].
Results S y n t h e s i s o f the parental replicative form D N A in dna ÷ cells Upon infection to dna ÷ hosts such as E. coli C or H502, phage G4 singlestranded DNA was converted to replicative form DNA at temperature ranging from 30 to 37°C, and this conversion was insensitive to 25--30 pg/ml of chloramphenicol (Fig. 1), 50 pg/ml of nalidixic acid and 50 pg/ml of mitomycin C, even in uvr ÷ E. coli (data not shown). At 42--43°C, replication of G4 phage was blocked in dna* cells as well as in dna ts E. coli strains. At 40°C, the phage yield in E. coil C dna ÷ was only 3% of that at 30°C (unpublished observations). As shown in Fig. 1B, however, synthesis of parental replicative form DNA was unaffected at temperature up to 43°C. Furthermore, 200 pg/ml of rifampicin did not inhibit this reaction in dna ÷ cells, even at 43°C (Fig. 1D). Parental replicative f o r m D N A f o r m a t i o n in dna m u t a n t s When E. coli strain C-2309 (dnaA ts) was grown at 43°C or at 30°C for 1 h and infected with ~4C-labeled G4, the single-stranded DNA was quantitatively
152
.A
B
dnaA
dnaB
dnaC(D)
dnaE
'
'
'
1
'0
'o
x K
0
.C
EO n 3 u
D
~'
u v o 2
, p,
o
I0
20
30
40
Fraction
10 No.
20
30
.tO
o
10
20
30
40 Fraction
i 10
i 20
3O
4O
No.
Fig. 1. S u c r o s e g r a d i e n t s e d i m e n t a t i o n of G4 single-stranded and parental replicative form DNA formed in H 5 0 2 d n a + cells. V i r a l D N A e x t r a c t e d f r o m p h a g e G 4 p a r t i c l e s l a b e l e d w i t h [ 14 C ] t h y m i n e or bacteria i n f e c t e d w i t h t h e p h a g e w a s c e n t r i f u g e d in a n e u t r a l s u c r o s e g r a d i e n t as d e s c r i b e d . T h e d i r e c t i o n o f s e d i mentation is f r o m r i g h t t o l e f t . A , p h a g e G 4 s i n g l e - s t r a n d e d D N A , B, r e p l i c a t i v c f o r m D N A f o r m a t i o n at 4 3 ° C ; C, P a r e n t a l r e p l i c a t i v e f o r m D N A s y n t h e s ~ e d at 37'~C in t h e p r e s e n c e o f 2 5 p g / m l o f c h l o r a m p h e n i c o l ; D , c o n v e r s i o n o f p h a g e G 4 s i n g l e - s t r a n d e d t o r e p l i c a t i v e f o r m D N A at 4 3 ° C in t h e p r e s e n c e o f 2 0 0 ttg/ml of rifarapicin. F i g . 2. C o n v e r s i o n o f p h a g e G 4 s i n g l e - s t r a n d e d t o r e p l i c a t i v e f r o m D N A in d n a A , dnaB, d n a C ( D ) a n d d n a E m u t a n t s . C e l l s o f C - 2 3 0 7 dnaA, L D 3 1 2 dnaB, L D 3 3 2 d n a C ( D ) or L D 3 0 1 d n a E w e r e i n f e c t e d w i t h G4 l a b e l e d w i t h [ 1 4 C ] t h y m i n e , at 3 0 ° C (--~)--) a n d 4 3 ° C ( - - $ - - ) . T h e viral D N A w a s e x t r a c t e d f r o m the infected bacteria and subjected to sedimentation a n a l y s i s as d e s c r i b e d . S e d i m e n t a t i o n is t o w a r d s t h e left.
converted to replicative form DNA at either temperature (Fig. 2). This result is in accord with the fact that none of the icosahedral single-stranded DNA phages hitherto tested (including $13, ¢R and a 3) required host DNA-initiation functions. In LD312 dnaB mutant (thermosensitive in DNA elongation), synthesis of the parental replicative form DNA occurred normally at 43 ° C. Similarly, host dnaC(D) mutation did not prevent conversion of phage G4 singlestranded to replicative form DNA. Furthermore, the infecting single-stranded DNA was converted to replicative form DNA at 43°C, in LD301 dnaE cells. Growth of these strains of bacteria did not occur at non-permissive temperatures. It seems noteworthy that conversion of phage ¢X174 single-stranded into replicative form DNA was unaffected in this DNA polymerase III mutant [7] as well as in strain H10261 p o l A p o l B dnaE [18], at nonpermissive temperatures. In contrast, formation of phage G4 parental RF in strain C-2309, a dnaG ts mutant, was restricted at 43°C, but not at 30°C (Fig. 3). In this dnaG t~ strain, defective in chain elongation, conversion of a3 single-stranded to parental replicative form DNA was also thermosensitive (unpublished result). Synthesis of phage G4 parental replicative form DNA was, like that of ~X174 parental replicative form DNA [19], not impaired at 43°C in HF4704S dnaH bacteria. The host dnaZ gene product is essential for replication of all single-
153 stranded DNA phages thus far studied. For instance, growth of phage ~bXtB, CKh-1 or ~3 in C727 dnaZ bacteria was thermosensitive at 43 ° C. Similarly, the phage yield in strain AX727 (dnaZ) transfected with single-stranded or replicative form DNA of phage S13 and ~R was markedly reduced at 43°C, compared with the yield of phage formed at 30°C {unpublished observation). As illustrated in Fig. 3, conversion of phage G4 single-stranded into replicative form DNA was completely prevented by the dnaZ mutation. When dnaZ ts cells were mixed with phage G4 at 30°C for 5 min and the mixture then shifted to 43 ° C, considerable parental replicative form DNA was already detectable in the infected bacteria. When the temperature shift was carried out after 10 min at 30°C, after infection, normal amounts of parental replicative form DNA were formed. Conversion o f single-stranded into replicative form DNA in E. coli rep- strain Although the rep function is not required for growth of E. coli cells, it is
required for replication of various single-stranded DNA phages such as ~X174, $13, OR, G13, G14, a3 and CKh-1 (unpublished data). The multiplication of G4 phage also depends on host rep product [1]. As seen in Fig. 3, synthesis of phage G4 parental replicative form DNA was not blocked by the rep mutation. On the other hand, no plaque was formed in D43 rep bacteria treated with CaCI2 and exposed to phage G4 replicative form DNA (unpublished observation). These results imply that replication of progeny replicative form DNA and/or single-stranded DNA depends on the rep gene product. In phage ~bX174 infection, synthesis of progeny replicative form DNA, but not parental replicative form DNA, is abortive in the rep strain [13].
clnaG
'
'
'
dn'aH
'
'
1
b K E 0
~3
_ clnaZ
.~
rep
,~io
2
10
20
30
~
40
10
20
30
40
Fraction No. F i g . 3. Parental replicative form D N A synthesis in dnaG, dnaH, d n a Z a n d rep m u t a n t s . Cells o f C - 2 3 0 9
dnaG, H F 4 7 0 4 S
dnaH, C 7 2 7 dnaZ o r D 4 3 r e p w e r e i n f e c t e d w i t h t h e l a b e l e d p h a g e G 4 at 3 0 ° C (- -o- -) o r
43°C ( e--). T h e i n f e c t e d b a c t e r i a w e r e l y z e d a n d v i r a l D N A w a s e x t r a c t e d as d e s c r i b e d . A n a l y s i s o f the DNA was performed by neutral sucrose velocity sedimentation. T h e d i r e c t i o n o f s e d i m e n t a t i o n is from fight to left.
154 Discussion Under the conditions employed, infecting phage G4 single-stranded DNA was converted to RF in strains thermosensitive in gene products dnaA, dnaB, dnaC(D), dnaE, dnaH or rep. The present data, however, demonstrate the involvement of dnaG and dnaZ gene products in the synthesis of G4 parental RF in vivo. Requirement of the dnaG protein has also been shown in phage G4 single-stranded to replicative form DNA conversion in vitro [5]. These data are consistent with the fact that synthesis of phage G4 parental replicative form DNA in dna ÷ cells is insensitive to 200 pg/ml of rifampicin. According to Zechel et al. [5], DNA unwinding protein and DNA polymerase III holoenzyme are also required for the conversion of phage G4 single-stranded to replicative form DNA. The role of DNA binding protein in the conversion of singlestranded phage G4 to replicative form in vivo is unknown; to date, there are no mutants known which affect formation of this protein. Occurrence of parental replicative form DNA synthesis in LD301 dnaE m u t a n t apparently suggests that, in contrast with the requirement in vitro, DNA polymerase III activity is dispensable in vivo. This might, however, be due to leakiness of the mutation in the particular strain employed. Another possibility is replacement of the DNA polymerase III function with DNA polymerase I and/or II. In order to discriminate between these possibilities, further experiments are in progress. Requirement of the dnaZ product is not elucidated in vitro in the G4 system [ 5]. On the other hand, Wickner and Hurwitz have more recently demonstrated involvement of the clnaZ protein (DNA elongation factor II) in the conversion in vitro of phages ~X174, fd and St-1 SS to duplex DNA [20,21]. Replicative form DNA synthesis in dnaZ cells infected with phage M13 or qSX174 is thermosensitive [12,22]. In addition, conversion of phage a 3 single-stranded to replicative form DNA in vivo depends upon the dnaZ gene product (manuscript in preparation). These data clearly indicate the importance of the dnaZ product in DNA replication. After completion of this work, Derstine et al. [23] reported that phage G4 trl was converted to the parental replicative form DNA at the nonpermissive temperature, in bacteria harboring dnaA, dnaB, dnaC(D), dnaE, or dnaG gene. Thus, their result with clnaG contradicts our results as well as those of Zechel et al. [ 5]. Whether this discrepancy is due to differences of phage strains (original phage G4 versus temperature-insensitive mutant) or not is unknown. In addition, the effect of the dnaZ mutation has not yet been tested for the phage G4 mutant. It is, however, quite probable that the clnaZ gene product may be required for the synthesis of phage G4 trl replicative form DNA as well. Acknowledgements We wish to thank Drs. R. Calendar, D.T. Denhardt, L.B. Dumas, G.N. Godson, T. Komano and J.R. Walker for providing bacterial strains and phage. References 1 T a k e t o , A. ( 1 9 7 5 ) P r o c e e d i n g s of the 1 9 7 5 M o l e c u l a r Biology M e e t i n g of ,Japan, pp. 118-.-120, K y o r i t s u S h u p p a n Co., T o k y o
155 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Taketo, A. (1976) Mol. Gen. Genet. 148, 139--142 Sinsheimer, R.L. (1968) Prog. Nucleic Acid Res. Mol. Biol. 8, 115--169 Denhardt, D.T. (1975) CRC Critical Rev. Microbiol. 4, 161--223 Zechel, K., Bouch~, J.-P. and Kornberg, A. (1975) J. Biol. Chem. 250, 4684---4689 Godson, G.N. (1974) Virology, 58, 272--289 Dumas, L.B. and Miller, C.A. (1973) J. Virol. 1 1 , 8 4 8 - - 8 5 5 Bowden, D.W., Twersky, R.S. and Calendar, R. (1975) J. Bacteriol. 124, 167--175 Dumas, L.B. and Miller, C.A. (1974) J. Virol. 14, 1369--1379 Dumas, L.B., Miller, C.A. and Bayne, M.L. (1975) J. Virol. 16, 575--580 Sakai, H., Hashimoto, S. and Komano, T. (1974) J. Bacteriol. 119, 811--820 Haldenwang, W.G. and Walker, J.R. (1977) J. Virol., in the press Denhardt, D.T., lwaya, M. and Larison, L.L. (1972) Virology 4 9 , 4 8 6 - - 4 9 6 Taketo, A. and Kuno, S. (1972) J. Biochem. 7 1 , 4 9 7 - - 5 0 5 Taketo, A. (1975) Z. Naturforsch. 30c, 800--803 Taketo, A. (1974) J. Biochem. 75, 895--904 Taketo, A. (1974) J. Biochem. 7 5 , 9 5 1 - - 9 6 0 Denhardt, D.T., Iwaya, M., McFadden, G. and Schochetman, G. (1973) Can. J. Biochem. 51, 1588--1597 Sakai, H. and Komano, T. (1975) Biochim. Biophys. Acta 395, 433--445 Wiekner, S. and Hurwitz, J. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1053--1057 Wickner, S. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3 5 1 1 - - 3 5 1 5 Haldenwang, G. and Walker, J.R. (1976) Biochem. Biophys. Res. Commun. 70, 932--938 Derstine, P.L., Dumas, L.B. and Miller, C.A. (1976) J. Virol. 1 9 , 9 1 5 - - 9 2 4
Biochimica et Biophysica Acta, 476 (1977) 149--155 © Elsevier/North-Holland Biomedical Press
BBA 98928 CONVERSION OF BACTERIOPHAGE G4 SINGLE-STRANDED VIRAL DNA TO DOUBLE-STRANDED REPLICATIVE FORM IN dna MUTANTS OF ESCHERICHIA COLI
KEN-ICHI K O D A I R A
and A K I R A
TAKETO
*
Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920 (Japan) (Received December 7th, 1976)
Summary Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escherichia coli. In dna ÷ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 #g/ml of rifampicin or 25 pg/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thevmosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43°C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.
Introduction Various enzymes and regulatory proteins necessary for host DNA synthesis are required for replication of single-stranded DNA phages as well. Thus, DNA polymerase III (dnaE gene product), the dnaG gene product, dnaZ protein, ribonucleotide reductase (nrdA gene product), and DNA ligase (lig gene product) are involved in replication of the St-1 group of phages including q~K [1], CXtB [2], and a3 (unpublished result). In addition to these functions, dnaB and dnaC(D) products are indispensable for replication of ¢X174 phage and other phages such as CA, G6, CB [1], S13 and ¢R (unpublished observation). Furthermore, all these phages require the rep function which is not required for replication of host bacteria. Replication of single-stranded (SS) DNA phages consists of three steps: conversion of single-stranded to parental replicative * To wh om requests for reprints s h o u l d b e sent.
150 form RF(I), subsequent replication of progeny RF(II) and asymmetrical synthesis of SS (III) [3]. In the case of ~X174, biochemical and genetic mechanisms operative in each step have been clarified considerably [4]. Recently, Zechel et al. [5] have reported that conversion of phage G4 DNA to RF II in vitro depends on three host proteins i.e dnaG protein, DNA binding protein and DNA polymerase III holoenzyme (which is known to be a complex of four discrete proteins). Regardless of the apparent simplicity of step I reaction in vitro, the host factor requirement in vivo is u n k n o w n for bacteriophage G4. The permissive growth temperature of phage G4 {phages G13, G14 and U3 as well) is relatively low and, at 42--43°C viral multiplication is severely restricted even in dna ÷ cells (unpublished observation). The enzymatic reaction involved in step I, however, occurs at the temperatures. In this report we have determined the host genetic factors required for synthesis of the parental replicative form in vivo. Materials and Methods Phage and bacterial strains. Bacteriophage G4 [6] was generously furnished by Dr. G.N. Godson. Escherichia coli H502 dna ÷ [7], C-2307 dnaA [8] and C2309 dnaG [8] were kindly provided by Dr. R. Calendar, strains LD312 dnaB [9], LD332 dnaC(D) [10] and LD301 dnaE [7] by Dr. L.B. Dumas, HF4704S dnaH [11] by Dr. T. Komano, C727 dnaZ [12] by Dr. J.R. Walker and strain D43 rep [13] by Dr. D.T. Denhardt. Preparation o f ~4C-labeled G4. Cells of E. coli H502 t hy- growing exponentially in TCG * medium [14] s lpplemented with 2 pg/ml of thymine were collected, washed, and resuspended in 50 ml of TCG medium at a density of A660 = 0.75. To the culture, 0.5 ml of 1 M CaC12, 1.5 • l 0 s plaque-forming units of phage G4, and 0.05 mCi of [~4C]thymine were added and incubation was continued at 37°C, with shaking. After lysis, EDTA was added to 20 mM and the lysate was centrifuged at 40 000 rev./min for 1 h. From the sediment, phages were extracted with 0.5% sodium dodecyl sulfate in 0.1 M NaCl/0.05 M Tris • HC1/0.01 M EDTA (pH 7.5), and subjected to several cycles of centrifugation at 14 000 rev./min and 40 000 rev./min. After treatment with 10 pg/ml of DNAase at 37°C for 1 h, the phages were layered on a 5 ml of 5--20% sucrose density gradient in dilution fluid [15] and centrifuged in a Hitachi RPS-50 rotor at 35 000 rev./min for 35 min. The gradient was collected dropwise from the b o t t o m and assayed for both ['4C] and plaque formation. The peak fractions were pooled, diluted with nutrient broth and stored at 4°C. The specific activity of the purified phage was 2 • 10 -6 cpm per plaque-forming unit. Infection. Bacteria were grown at 30°C in nutrient broth, with shaking, to a density of A6~o = 0.25 and divided into two 10 ml portions. One portion was shifted to 43°C and the other kept at 30°C. Each culture received 0.1 ml of 1 M CaC12 and, 10 min after the temperature shift, infected with ~4C-labeled phage G4 at a multiplicity of four. When initiation-defective dnaA or dnaH mutants of E. coli were infected, cells were pregrown at 43°C for 1 h. The infected culture was incubated for 15 min at 30°C or for 20 min at 43°C, with shaking. • TCG, Tris]casamino acids[~lucose.
151 Cell lysis and D N A extraction. Infection was terminated by pouring each culture into an equal volume of ice-cold 75% ethanol in 0.05 M Tris • HC1/0.02 M EDTA (pH 8.0). The cells were harvested and washed with chilled 0.05 M Tris • HC1/0.01 M EDTA (pH 8.0) by a brief centrifugation and suspended in the buffer. Lysozyme was added to a final concentration of 0.25 mg/ml and the mixture was kept at 0°C for 10 min and then at room temperature for 10 min. After addition of 0.5% sodium dodecyl sulfate, the mixture was incubated for 30 min at room temperature and treated further with 0.5 mg/ml of autodigested pronase for 4.5 h at 37°C. The sample was subsequently treated with an equal volume of 90% phenol to extract nucleic acids. After removal of residual phenol with ether, NaC1 was added to a final concentration of 1 M and the mixture was allowed to stand for 2 h at 0°C. The precipitated bacterial DNA was removed by a low speed centrifugation and the supernatant was subjected to sedimentation analysis. Analysis o f D N A on sucrose gradients. The DNA sample (0.1 ml) was layered onto a 5 ml of 5--20% sucrose density gradient in 0.05 M Tris • HCI/0.002 M EDTA/1 M NaC1 (pH 7.2) and centrifuged in a Hitachi RPS-50 rotor at 40 000 rev./min for 4 h, at 4°C. Fractions were collected from the b o t t o m of the gradient. D e t e r m i n a t i o n o f radioactivity. Aliquots from sucrose gradient fractions were spotted on glass filter paper, dried, and counted in a Nuclear Chicago liquid scintillation spectrometer. The scintillation fluid contained, per liter of toluene, 4 g of 2,5-diphenyloxazole and 0.1 g of 1.4-bis(2-(5-phenyloxazolyl))benzene. Others. [~4C]Thymine (58 Ci/mol) was purchased from the Radiochemical Centre. Rifampicin was obtained from Daiichi Pharmaceutical Co. Tokyo. Phage G4 single-stranded DNA was extracted from the purified phage by phenol method. The sources of other materials were as previously described [16,17].
Results S y n t h e s i s o f the parental replicative form D N A in dna ÷ cells Upon infection to dna ÷ hosts such as E. coli C or H502, phage G4 singlestranded DNA was converted to replicative form DNA at temperature ranging from 30 to 37°C, and this conversion was insensitive to 25--30 pg/ml of chloramphenicol (Fig. 1), 50 pg/ml of nalidixic acid and 50 pg/ml of mitomycin C, even in uvr ÷ E. coli (data not shown). At 42--43°C, replication of G4 phage was blocked in dna* cells as well as in dna ts E. coli strains. At 40°C, the phage yield in E. coil C dna ÷ was only 3% of that at 30°C (unpublished observations). As shown in Fig. 1B, however, synthesis of parental replicative form DNA was unaffected at temperature up to 43°C. Furthermore, 200 pg/ml of rifampicin did not inhibit this reaction in dna ÷ cells, even at 43°C (Fig. 1D). Parental replicative f o r m D N A f o r m a t i o n in dna m u t a n t s When E. coli strain C-2309 (dnaA ts) was grown at 43°C or at 30°C for 1 h and infected with ~4C-labeled G4, the single-stranded DNA was quantitatively
152
.A
B
dnaA
dnaB
dnaC(D)
dnaE
'
'
'
1
'0
'o
x K
0
.C
EO n 3 u
D
~'
u v o 2
, p,
o
I0
20
30
40
Fraction
10 No.
20
30
.tO
o
10
20
30
40 Fraction
i 10
i 20
3O
4O
No.
Fig. 1. S u c r o s e g r a d i e n t s e d i m e n t a t i o n of G4 single-stranded and parental replicative form DNA formed in H 5 0 2 d n a + cells. V i r a l D N A e x t r a c t e d f r o m p h a g e G 4 p a r t i c l e s l a b e l e d w i t h [ 14 C ] t h y m i n e or bacteria i n f e c t e d w i t h t h e p h a g e w a s c e n t r i f u g e d in a n e u t r a l s u c r o s e g r a d i e n t as d e s c r i b e d . T h e d i r e c t i o n o f s e d i mentation is f r o m r i g h t t o l e f t . A , p h a g e G 4 s i n g l e - s t r a n d e d D N A , B, r e p l i c a t i v c f o r m D N A f o r m a t i o n at 4 3 ° C ; C, P a r e n t a l r e p l i c a t i v e f o r m D N A s y n t h e s ~ e d at 37'~C in t h e p r e s e n c e o f 2 5 p g / m l o f c h l o r a m p h e n i c o l ; D , c o n v e r s i o n o f p h a g e G 4 s i n g l e - s t r a n d e d t o r e p l i c a t i v e f o r m D N A at 4 3 ° C in t h e p r e s e n c e o f 2 0 0 ttg/ml of rifarapicin. F i g . 2. C o n v e r s i o n o f p h a g e G 4 s i n g l e - s t r a n d e d t o r e p l i c a t i v e f r o m D N A in d n a A , dnaB, d n a C ( D ) a n d d n a E m u t a n t s . C e l l s o f C - 2 3 0 7 dnaA, L D 3 1 2 dnaB, L D 3 3 2 d n a C ( D ) or L D 3 0 1 d n a E w e r e i n f e c t e d w i t h G4 l a b e l e d w i t h [ 1 4 C ] t h y m i n e , at 3 0 ° C (--~)--) a n d 4 3 ° C ( - - $ - - ) . T h e viral D N A w a s e x t r a c t e d f r o m the infected bacteria and subjected to sedimentation a n a l y s i s as d e s c r i b e d . S e d i m e n t a t i o n is t o w a r d s t h e left.
converted to replicative form DNA at either temperature (Fig. 2). This result is in accord with the fact that none of the icosahedral single-stranded DNA phages hitherto tested (including $13, ¢R and a 3) required host DNA-initiation functions. In LD312 dnaB mutant (thermosensitive in DNA elongation), synthesis of the parental replicative form DNA occurred normally at 43 ° C. Similarly, host dnaC(D) mutation did not prevent conversion of phage G4 singlestranded to replicative form DNA. Furthermore, the infecting single-stranded DNA was converted to replicative form DNA at 43°C, in LD301 dnaE cells. Growth of these strains of bacteria did not occur at non-permissive temperatures. It seems noteworthy that conversion of phage ¢X174 single-stranded into replicative form DNA was unaffected in this DNA polymerase III mutant [7] as well as in strain H10261 p o l A p o l B dnaE [18], at nonpermissive temperatures. In contrast, formation of phage G4 parental RF in strain C-2309, a dnaG ts mutant, was restricted at 43°C, but not at 30°C (Fig. 3). In this dnaG t~ strain, defective in chain elongation, conversion of a3 single-stranded to parental replicative form DNA was also thermosensitive (unpublished result). Synthesis of phage G4 parental replicative form DNA was, like that of ~X174 parental replicative form DNA [19], not impaired at 43°C in HF4704S dnaH bacteria. The host dnaZ gene product is essential for replication of all single-
153 stranded DNA phages thus far studied. For instance, growth of phage ~bXtB, CKh-1 or ~3 in C727 dnaZ bacteria was thermosensitive at 43 ° C. Similarly, the phage yield in strain AX727 (dnaZ) transfected with single-stranded or replicative form DNA of phage S13 and ~R was markedly reduced at 43°C, compared with the yield of phage formed at 30°C {unpublished observation). As illustrated in Fig. 3, conversion of phage G4 single-stranded into replicative form DNA was completely prevented by the dnaZ mutation. When dnaZ ts cells were mixed with phage G4 at 30°C for 5 min and the mixture then shifted to 43 ° C, considerable parental replicative form DNA was already detectable in the infected bacteria. When the temperature shift was carried out after 10 min at 30°C, after infection, normal amounts of parental replicative form DNA were formed. Conversion o f single-stranded into replicative form DNA in E. coli rep- strain Although the rep function is not required for growth of E. coli cells, it is
required for replication of various single-stranded DNA phages such as ~X174, $13, OR, G13, G14, a3 and CKh-1 (unpublished data). The multiplication of G4 phage also depends on host rep product [1]. As seen in Fig. 3, synthesis of phage G4 parental replicative form DNA was not blocked by the rep mutation. On the other hand, no plaque was formed in D43 rep bacteria treated with CaCI2 and exposed to phage G4 replicative form DNA (unpublished observation). These results imply that replication of progeny replicative form DNA and/or single-stranded DNA depends on the rep gene product. In phage ~bX174 infection, synthesis of progeny replicative form DNA, but not parental replicative form DNA, is abortive in the rep strain [13].
clnaG
'
'
'
dn'aH
'
'
1
b K E 0
~3
_ clnaZ
.~
rep
,~io
2
10
20
30
~
40
10
20
30
40
Fraction No. F i g . 3. Parental replicative form D N A synthesis in dnaG, dnaH, d n a Z a n d rep m u t a n t s . Cells o f C - 2 3 0 9
dnaG, H F 4 7 0 4 S
dnaH, C 7 2 7 dnaZ o r D 4 3 r e p w e r e i n f e c t e d w i t h t h e l a b e l e d p h a g e G 4 at 3 0 ° C (- -o- -) o r
43°C ( e--). T h e i n f e c t e d b a c t e r i a w e r e l y z e d a n d v i r a l D N A w a s e x t r a c t e d as d e s c r i b e d . A n a l y s i s o f the DNA was performed by neutral sucrose velocity sedimentation. T h e d i r e c t i o n o f s e d i m e n t a t i o n is from fight to left.
154 Discussion Under the conditions employed, infecting phage G4 single-stranded DNA was converted to RF in strains thermosensitive in gene products dnaA, dnaB, dnaC(D), dnaE, dnaH or rep. The present data, however, demonstrate the involvement of dnaG and dnaZ gene products in the synthesis of G4 parental RF in vivo. Requirement of the dnaG protein has also been shown in phage G4 single-stranded to replicative form DNA conversion in vitro [5]. These data are consistent with the fact that synthesis of phage G4 parental replicative form DNA in dna ÷ cells is insensitive to 200 pg/ml of rifampicin. According to Zechel et al. [5], DNA unwinding protein and DNA polymerase III holoenzyme are also required for the conversion of phage G4 single-stranded to replicative form DNA. The role of DNA binding protein in the conversion of singlestranded phage G4 to replicative form in vivo is unknown; to date, there are no mutants known which affect formation of this protein. Occurrence of parental replicative form DNA synthesis in LD301 dnaE m u t a n t apparently suggests that, in contrast with the requirement in vitro, DNA polymerase III activity is dispensable in vivo. This might, however, be due to leakiness of the mutation in the particular strain employed. Another possibility is replacement of the DNA polymerase III function with DNA polymerase I and/or II. In order to discriminate between these possibilities, further experiments are in progress. Requirement of the dnaZ product is not elucidated in vitro in the G4 system [ 5]. On the other hand, Wickner and Hurwitz have more recently demonstrated involvement of the clnaZ protein (DNA elongation factor II) in the conversion in vitro of phages ~X174, fd and St-1 SS to duplex DNA [20,21]. Replicative form DNA synthesis in dnaZ cells infected with phage M13 or qSX174 is thermosensitive [12,22]. In addition, conversion of phage a 3 single-stranded to replicative form DNA in vivo depends upon the dnaZ gene product (manuscript in preparation). These data clearly indicate the importance of the dnaZ product in DNA replication. After completion of this work, Derstine et al. [23] reported that phage G4 trl was converted to the parental replicative form DNA at the nonpermissive temperature, in bacteria harboring dnaA, dnaB, dnaC(D), dnaE, or dnaG gene. Thus, their result with clnaG contradicts our results as well as those of Zechel et al. [ 5]. Whether this discrepancy is due to differences of phage strains (original phage G4 versus temperature-insensitive mutant) or not is unknown. In addition, the effect of the dnaZ mutation has not yet been tested for the phage G4 mutant. It is, however, quite probable that the clnaZ gene product may be required for the synthesis of phage G4 trl replicative form DNA as well. Acknowledgements We wish to thank Drs. R. Calendar, D.T. Denhardt, L.B. Dumas, G.N. Godson, T. Komano and J.R. Walker for providing bacterial strains and phage. References 1 T a k e t o , A. ( 1 9 7 5 ) P r o c e e d i n g s of the 1 9 7 5 M o l e c u l a r Biology M e e t i n g of ,Japan, pp. 118-.-120, K y o r i t s u S h u p p a n Co., T o k y o
155 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Taketo, A. (1976) Mol. Gen. Genet. 148, 139--142 Sinsheimer, R.L. (1968) Prog. Nucleic Acid Res. Mol. Biol. 8, 115--169 Denhardt, D.T. (1975) CRC Critical Rev. Microbiol. 4, 161--223 Zechel, K., Bouch~, J.-P. and Kornberg, A. (1975) J. Biol. Chem. 250, 4684---4689 Godson, G.N. (1974) Virology, 58, 272--289 Dumas, L.B. and Miller, C.A. (1973) J. Virol. 1 1 , 8 4 8 - - 8 5 5 Bowden, D.W., Twersky, R.S. and Calendar, R. (1975) J. Bacteriol. 124, 167--175 Dumas, L.B. and Miller, C.A. (1974) J. Virol. 14, 1369--1379 Dumas, L.B., Miller, C.A. and Bayne, M.L. (1975) J. Virol. 16, 575--580 Sakai, H., Hashimoto, S. and Komano, T. (1974) J. Bacteriol. 119, 811--820 Haldenwang, W.G. and Walker, J.R. (1977) J. Virol., in the press Denhardt, D.T., lwaya, M. and Larison, L.L. (1972) Virology 4 9 , 4 8 6 - - 4 9 6 Taketo, A. and Kuno, S. (1972) J. Biochem. 7 1 , 4 9 7 - - 5 0 5 Taketo, A. (1975) Z. Naturforsch. 30c, 800--803 Taketo, A. (1974) J. Biochem. 75, 895--904 Taketo, A. (1974) J. Biochem. 7 5 , 9 5 1 - - 9 6 0 Denhardt, D.T., Iwaya, M., McFadden, G. and Schochetman, G. (1973) Can. J. Biochem. 51, 1588--1597 Sakai, H. and Komano, T. (1975) Biochim. Biophys. Acta 395, 433--445 Wiekner, S. and Hurwitz, J. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1053--1057 Wickner, S. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3 5 1 1 - - 3 5 1 5 Haldenwang, G. and Walker, J.R. (1976) Biochem. Biophys. Res. Commun. 70, 932--938 Derstine, P.L., Dumas, L.B. and Miller, C.A. (1976) J. Virol. 1 9 , 9 1 5 - - 9 2 4