Molec. gen. Genet. 135, 73--86 (1974) © by Springer-Verlag 1974

In vitro Studies on the Role of Phage T7 Gene 4 Product in DNA Replication Eberhard Scherzinger and Frank Litfin Max-Planck-Institut ffir Molekulare Genetik, Abteilung Schuster, Berlin-Dahlem, Germany Received October 23, 1974 Summary. A soluble enzyme fraction prepared from T7-infected E. coli is able to initiate DNA synthesis on circular single-stranded phage DNA. The product synthesized in vitro is a full-length linear complementary strand as judged by alkaline sucrose gradient analysis. DNA synthesis requires the products of the phage genes 4 and 5, Mg++, dNTPs and rNTPs; however, ATP by itself can almost completely satisfy the rNTP requirement. The gene 4 product is essential for DNA chain initiation on unprimed single-stranded DNA, but is dispensable for the replication of a ¢X174 DNA-RNA hybrid. The enzyme system from T7-infected cells does not discriminate between the I)NA templates from phages ¢X174, M13 or fd and is also capable of replicating native T7 DNA. However, a striking difference with regard to the template DNA is revealed by eomplementation analysis. Extracts of T7 mutantinfected cells complement each other only with T7 DNA but not with ¢X174 DNA as template. Abbreviations: rNTP ribonucleoside triphosphate; dNTP deoxyribonucleoside triphosphate; BSA bovine serum albumin.

Introduction Recently Str/ttling etal. (1973) and Itinkle and Richardson (1974) have developed in vitro systems from phage T7-infected E. coli which are capable of replicating exogenous duplex T7 DNA. DNA synthesis in these crude i n vitro systems closely mimics T7 DNA replication i n vivo. The in vitro reaction is dependent on the phage gene 4 and gene 5 products. While the gene 5 protein has been identified as T7 DNA polymerase (Grippo and Richardson, 1971; Oey et al., 1971), the enzymatic activity associated with the gene 4 protein has still to be elucidated. I n this communication we describe experiments which may lead to a better understanding of the role of the gene 4 product in DNA replication. We found that a soluble enzyme fraction extracted from T7-infected E. coli is capable of converting circular single-stranded dpX174 DNA to duplex structures. This relatively simple replicative operation requires both T7 DNA polymerase and the gene 4 product. We provide evidence that in this system the gene 4 protein acts at the stage of DNA chain initiation. Materials and Methods Materials Lysozyme, ribonucleoside and deoxyribonucleoside triphosphates obtained from Boehringet Mannheim GmbH (Mannheim), radioactively labeled nucleotides from Radiochemicals (Amersham, U. K.), a, fl-methylene- and fl, y-methylene-adenosine triphosphate from Miles Chemical Company, rifampicin and actinomycin D from Calbiochem and nitrocellulose filters from Membranfilter GmbH (G5ttingen).

74

E. Scherzinger and F. Litfin

Bacteria and Bacteriophages T7 amber m u t a n t s used in this work were: T7 H668 (am gene 4) from Dr. R. H a u s m a n n ; T7 am29 (am gene 3), T7 am28 (am gene 5), and T7 am147 (am gene 6) from Dr. F. W. Studier. The T7 double and triple m u t a n t s used in this study were constructed in our laboratory and, for convenience, are designed b y the gene n u m b e r only. Phage crosses and complementation tests were performed as described b y Studier (1969). The cell extracts were prepared from TT-infected E. coli H559 (pol A 1, end A, F-, fly-, lac-, su-). This strain was kindly supplied b y Dr. H. Hoffmann-Berling.

Enzymes Purified T7 R N A polymerase was prepared as previously described (Scherzinger et al., 1973). E. coli R N A polymerase was isolated by the method of Burgess (1969).

D N A Templates • Unlabeled a n d [14C] labeled phage ¢X174 DNA was prepared b y a minor modification of the method of Francke and R a y (1971). Phage M13 a n d fd D N A were kindly provided b y Dr. K. Geider, Heidelberg. RNA-primed ¢X174 DNA was prepared with E. coli R N A polymerase according to Wiekner et al. (1973). T7 DNA was extracted with phenol from purified phage particles as described b y Center and Richardson (1970). Heat-denatured calf t h y m u s DNA was prepared as described b y Grippo and Richardson (1971).

Preparation o] Extracts A one-liter culture of E. coli H559 was grown at 37°C in M9-medium (Studier, 1969) supplemented with 1.5% casamino acids and 20 ~g thymine/ml. A t a cell density of 4 × 108/ml, cells were pelleted in the cold and resnspended in M9 (without Mg++) a t a cell concentration of 1 × 101°/ml. T7 phages were added a t a multiplicity of 10. The phages were allowed to adsorb at 10 ° C for 3 rain. The infected cells were t h e n diluted into 500 ml ~¢I9-medium prewarmed to 30 ° C. After 15 rain a t 30 ° C, the culture was rapidly chilled to 4 ° C. The cells were harvested b y centrifugation at 4 ° C, washed once with 50 mM Tris-HC1 (pH 7.6)--0.1 M NaC1 a n d resuspended in 3 ml 0.1 M Tris-HC1 (pH 7.6). The cell suspension was frozen in a dry icemethanol b a t h and stored a t -- 25 ° C. Frozen cells were thawed a t room temperature. Sucrose (0.6 ml of a 60% solution) a n d lysozyme (0.4 ml, 2 mg/ml in 0.25 M Tris-HCl--1 mM EDTA, p H 7.6) were added. After incubation at 4 ° C for 45 min, 1 ml 5 IV[ NaC1 was added a n d the lysato was centrifuged a t 4 ° C for 2 h at 40000 rev./min in a 50 Ti-rotor. To the t r a n s p a r e n t s u p e r n a t a n t 2 volumes of a neutralized s a t u r a ~ d ammonium-sulfate solution were added. After stirring a t 4°C for 30 rain, the precipitate was collected b y centrifugation and redissolved in 4 ml 10% (w/v) sucrose---20 mM Tris-HCl (pH 7.6)--0.1 mM E D T A - - l m M dithioerythritol--0.2 M KC1 (buffer A). The protein solution was dialysed overnight against two changes of 500 ml of buffer A. The a m m o n i u m sulfate fractions thus prepared h a d a ratio A2s0:A2~0 of a b o u t 0.65 and contained 12-13 mg protein/ml. The cell extracts could be stored frozen a t -- 25 ° C for 2 weeks without detectable loss of DNA synthesizing activity. Dilutions of cell extracts were made in buffer A containing 1 rag BSA/ml.

Standard A s s a y Condition8 Unless otherwise specified, the assay mixture (final volume 50 lzl) contained 40 ml~¢I Tris-HC1 (pH 7.6), 10 mM ~gC12, 5 mM dithi0erythritol, 0.25 m ~ each of rATP, rGTP, rCTP and rUTP, 50 tiM each of dATP, dGTP, dCTP and [aH]dTTP, 15 t~g rifampicin/ml, 0.5 mg BSA/ml, 3.4 nmoles ~X174 DNA and 10 iz! of a n a m m o n i u m sulfate fraction prepared as described above (final KC1 concentration was 40 raM). T h e reaction was started b y the addition of the enzyme fraction. A f t e r incubation at 30 ° C for the indicated time, the reaction was terminated b y the addition of 5 % trichloroacetic

Function of Phage T7 Gene & Product

750-

75

T73,6[1smlnl

500-

F- 250-

"rT~,~,6!~s~i.l~

/" 5

10

15

;rTj 61sm+i-,j 20

Time 30°C [mfn)

Fig. 1. Kinetics of ~X174 DNA-directed DNA synthesis. The time course of DNA synthesis was measured using the standard assay conditions described under "Methods ". The extracts were prepared from E. coli I-I559 infected with the indicated T7 amber mutants. Time after infection at which cells have been collected is given in parentheses acid containing 0.1 M sodium pyrophosphate. The precipitate was collected on nitrocellulose filters, washed twice with 5 % and finally with 2 % triehloroacetic acid. The filters were dried and counted in a toluene-based scintillation fluid.

Assay o] T7 D N A and R N A Polymerase Activity T7 DNA polymerase activity in extracts from T7.infected cells was measured in a reaction mixture (50 ~l) containing 40 mM Tris-HC1 (pH 7.6), 10 mM MgC12, 40 mM KCI, 50 FM each of the four dNTPs ([3tt]dTTP), 16 nmoles of heat-denatured calf thymus DNA, 0.5 mg BSA/ml, and 1 mX~ dithioerythritol. T7 R N A polymerase was assayed in an incubation mixture (50 tzl) containing 40 mM Tris-HC1 (pH 7.6), 20 mM MgC12, 40 mM KC1, 0.25 m3I each of the four rNTPs ([aH]rCTP), 3.4 nmoles T7 DNA, 20 ~zg rifampiein/ml, 1 mg BSA/ml, and 1 mM dithioerythritol. The reactions were started by the addition of 5 ~zl of a diluted extract to be tested for enzyme activity. After incubation for 10 min at 30 ° C, the acid-insoluble radioactivity was determined as described above.

Other Procedures Sucrose step gradients (20 to 5%) in 0.15 M KOH, 50 mM glycine, 0.4 M KC1 and 1 mM E D T A were prepared as described by Tabak et al. (1974). Sedimentation was for 4.5 h at 50000 rev./min in a SW 50 rotor at 5 ° C. Protein concentrations were determined by the method of Biicher (1947). DIqA concentrations were calculated from the reported specific absorbancies at 260 nm (Sinsheimer, 1966; Thomas and Abelson, 1966). Unless otherwise specified, DNA concentration is expressed as nueleotide equivalents. Results

CX17d DNA.directed D N A Synthesis in Extracts/rom T7,in]ected Cells E x t r a c t s p r e p a r e d f r o m E. coli t t 5 5 9 (pol A1) i n f e c t e d w i t h T7 c a r r y i n g a m b e r m u t a t i o n s in b o t h g e n e 3 ( e n d o n u c l e a s e ) a n d g e n t 6 (exonuclease) w e r e f o u n d to c a t a l y z e e x t e n s i v e dpX174 D N A - d i r e c t e d d e o x y n u e l e o t i d e i n c o r p o r a t i o n (Fig. 1).

76

E. Scherzingcr and F. Litfin Table 1. Requirements for (~X174 DNA-directed DNA synthesis in vitro Additions

Activity

T7a,6

T73,4,6

Complete q-Aetinomyein D (5 fzg/ml) --Rifampicin --DNA --Mg++-kEDTA (i mM)

100 86 106 <2 <1

10 -10 <2 <1

--dATP, dGTP, d C T P

< 1

< 1

%

--4rNTP --4rNTP-~rATP (1-2 raM) -brGTP (1 mM) -krCTP (1 toNI) -~rUTP (1 mM) -kApp(CH2)p (2 mM) -kAp(CH2)pp (2 mM)

10 78-85 41 36 35 5 6

%

7 9 11 8 7 5 7

Activity was determined as described under "Methods". In the complete system 530 pmoles [3H]TMP were incorporated in 10 rain at 30°C using extracts prepared from E.coli H559 infected with T7a,e or T73,4,6.

DNA synthesis was dependent on at least two phage gene products. With extracts lacking either the T7 gene 4 product, or the gene 5 product (DNA polymerase) the rate and extent of DNA synthesis was less than 10% of that observed with extracts from T78,s-infeeted cells. T7 amber mutants deficient in the gene 3 and 6 nucleases were used for infection to insure that no extensive breakdown of the DNA substrate occurred. Replication of ~X174 DNA by host-cell enzymes appeared not to occur under the conditions of our system, since with extracts from infected cells collected 5 min after infection essentially no synthesis was detected (Fig. 1). The crude extracts were routinely precipitated with ammonium sulfate at 60% saturation to remove low levels of endogenous nucleotides. Close to 100% of the DNA synthesizing activity in such extracts could be recovered in fractions precipitated between 30 and 60 % saturated ammonium sulfate. The recovery of activity per cell equivalent in a 35-60% ammonium sulfate fraction was still 70 % of that observed in an unfractionated extract.

Requirements/or D1VA Synthesis and Optimal Conditions The requirements for ¢~X174 DNA-directed DNA synthesis catalyzed by extracts from cells infected with either T7s, e or T7a,4, s mutants are shown in Table 1. The reaction was dependent on NIg++, the four dNTPs and added (~X174 DNA. With extracts from TTs,6-infected cells, omission of the four rNTPs resulted in a 10-fold reduction in DNA synthesizing activity. Extracts lacking the gene 4 product, however, showed no significant response to the addition of ribonncleoside triphosphates. Nearly maximal activity was obtained when the four rNTPs

Function of Phage T7 Gene 4 Product

77

Table 2. Template specificity for DNA synthesis Template DNA

Activity

fd M13 qbX174 RNA-primed (~X174 T7 Denatured calf thymus

T73,6 %

T73,4.6 %

100 92 as 90 18 50

27 26 16 75 2 38

Standard assay mixtures were used, except that the reactions contained 15 mM MgC12, 0.25 mM each of the four dNTPs and 3.4nmoles of the template DNA indicated. 100% activity was equivalent to the incorporation of 820 pmoles [3H]TMP in 10 rain at 30° C, using 10 [~l of extracts prepared from cells infected with T73,6 or T73,4,6.

were replaced by A T P alone, whereas any one of the other ribonucleoside triphosphates could only partially substitute for ATP. Neither ~, fi-methylenenor fl, y-methylene-adenosine triphosphate could replace ATP. Rifampiein, an inhibitor of E. coli R N A polymerase, had no effect on ~bX174 DNA-dependent DNA synthesis. Actinomyein D intercalates into helical DNA and specifically inhibits R N A synthesis (Sobell et al., i971). At low concentrations of this drug, D N A synthesis was not profoundly affected in our in vitro system. I n contrast, the different E. coli enzyme systems that are known to be required for initiation of D N A synthesis on dpX174 and M13 DNA are almost completely inhibited by low amounts of actinomycin (Schekman et al., 1972). With extracts prepared from T78,6-infected cells m a x i m u m rate of ~X174 DNA-dircctcd synthesis was obtained using Tris-HC1 buffer (40 raM) in the pHrange 7.5 to 7.8. The reaction was stimulated by the addition of KC1 up to 80 raM. At 0.15M KC1, the rate of DNA synthesis was 60% of the maximum. I n the presence of 80 mM KC1, the optimal Mg ++ concentration was 10 raM.

Template Speci]icity D N A synthesis was measured in extracts from T7~, 6- and T78,4,~-infected cells using various DNA templates (Table 2). After 10 rain of synthesis, the amount of DNA synthesized was roughly the same when circular single-stranded DNAs of either phage dpX174, M13 or fd were used, indicating that the gene 4 protein is not completely template specific. I n extracts lacking the gene 4 product, DATA synthesis was less than 30 % of that observed in extracts from T7a,s-infected cells. However, the gene 4 protein seems to be dispensable for the replication of single-stranded D N A containing a 3'-OH RNA or DNA primer end. Therefore, we conclude t h a t the gene 4 protein functions at the stage of DNA chain initiation. DNA synthesis using native T7 DNA as template was also dependent on the gene 4 product confirming the results reported by Str~tling and Knippers (1973) and Hinkle and Richardson (1974).

78

E. Scherzinger and F. Litfin

CX virat DNA

4-

2i -1 1-

lb

2b

a'o

Fraction Number

Fig. 2. Characterization of the product DNA synthesized in vitro. A reaction mixture (0.1 ml) containing 40 mM Tris-HC1 (pit 7.6), 15 mM MgC12, 5 mM dithioerythritol, 0.25 mM each of the four rNTPs, 0.25 mM each of the four dNTPs ([3H]dTTP), 15 ~zg rifampicin/ml, 0.5 mg BSA/ml, 3.4 nmoles ¢X174 [14C] DNA and 25 ~l of an extract prepared from T73.6-infected E. coli I-I559 was incubated for 10 min at 30 ° C. A total of 3.5 nmoles deoxynucleotide has been incorporated into acid-insoluble material as determined from an aliquot of the reaction mixture. To a 50-F1 aliquot an equal volume of a solution containing 2% Sarcosyl, 0.3 M KOH, 0.1 M glycine, 0.8 M KC1 and 40 mM EDTA was added. The mixture was incubated for 10 rain at 42 ° C and analyzed by sedimentation through an alkaline sucrose gradient as described in "Methods". A gradient containing [14C]-labeled ~X174 DNA was run in parallel to provide a sedimentation marker

Characterization o/the Product DNA D N A s y n t h e s i z e d in vitro in a r e a c t i o n m i x t u r e c o n t a i n i n g e x t r a c t f r o m T73, 6infected E. coli H559, [14C] dpX174 D N A as t e m p l a t e a n d [ 3 H ] d T T P was a n a l y z e d b y s e d i m e n t a t i o n t h r o u g h a n alkaline sucrose g r a d i e n t (Fig. 2): 1) T h e a m o u n t of n e w l y s y n t h e s i z e d D N A was e q u i v a l e n t to t h e a m o u n t of t e m p l a t e D N A added. 2) T h e m a j o r i t y of t h e t e m p l a t e D N A r e m a i n e d i n t a c t d u r i n g t h e incubation. 3) T h e p r o d u c t D N A s e d i m e n t e d to t h e p o s i t i o n of full-length linear dpX174 D N A (16 S). This result p o i n t s to a s t a r t of r e p l i c a t i o n from a u n i q u e site on t h e t e m p l a t e D N A r a t h e r t h a n to m u l t i p l e initiations.

E//ect o/Nucleoside TriphosThate Concentration The effects of v a r y i n g t h e c o n c e n t r a t i o n s of t h e four ribonueleoside triphosp h a t e s on t h e r a t e of D N A s y n t h e s i s are i l l u s t r a t e d in Fig. 3 A. I n e x t r a c t s containing t h e gene 4 p r o d u c t the r a t e of D N A synthesis could be s t i m u l a t e d as m u c h as 20-fold w i t h increasing r N T P c o n c e n t r a t i o n , whereas in e x t r a c t s lacking t h e gene 4 p r o d u c t r N T P c o n c e n t r a t i o n s u p t o 0.5 m M h a d o n l y v e r y l i t t l e s t i m u l a t o r y effect on t h e r a t e of synthesis. The a p p a r e n t K m for t h e s e s u b s t r a t e s was a b o u t 0.15 mM.

Function of Phage T7]Gene 4 Product

~(B)

-- I {A)

i~ 601 !

~

79

T a /e,'

T'73'6

4o T73,~ ~: 2o

0J 0.2 03 0.£ 0.5 [rNTP](rnM] i

i

L

]

p

0.1 (12 0.3 0.4 0.5 [dNTP](rnN)

Fig. 3A and B. Effect of ribonueleoside and deoxynucleoside triphosphate concentration on the rate of DNA synthesis. Reactions were performed as described under "Methods", except that the concentrations of each of the four rNTPs or dNTPs were varied as indicated. The average rates of synthesis were determined from a 2- and 4-min incubation at 30° C. (A) dNTP concentration was kept at 50 FM each. (B) Each of the four rNTPs was present at 0.25 mM (solid line), or the four rNTPs have been omitted (dotted line)

The effect of deoxynucleoside triphosphate concentration on the initial rate of D N A synthesis is shown in Fig. 3 B. I n the presence of ribonueleoside triphosphates (0.25 mlV[ each), half maximal rate of DIXTA synthesis in an extract from T7a,8-infected cells was obtained when each one of the four d N T P s was present at 50 ~zM. Omission of the ribonncleoside triphosphates reduced the rate of synthesis to anywhere between 5 and 30%, depending on the d N T P concentration. The unusually high K m for d N T P s in this case m a y be explained b y contaminations of the deoxynucleoside triphosphates with low levels of ribonucleoside triphosphates. I n extracts from T73,a,~-infected cells, the residual D N A synthesis was not affected b y ribonucleoside triphosphates at all d N T P concentrations tested.

E]]ect o] Extract and Template DNA Concentration The rate of dpX174 DNA-directed D N A synthesis was determined in 50-F1 s t a n d a r d reaction mixtures, varying the concentrations of extracts prepared from T73, 6- and T73,4,6-infected cells (Fig. 4). Using extract a m o u n t s up to 2.5 tzl per assay, the rate of D N A synthesis was indistinguishable in extracts prepared from T78,G- or T7s,4,6-infected cells. However, increasing the a m o u n t of extract up to 20 F1 led to a striking stimulation of D N A synthesis in T73,~-extraets, whereas there was no additional stimulatory effect in extracts lacking the gene 4 product. At low extract concentrations, the rate of D N A synthesis was not proportional to the a m o u n t of extract added. Most p r o b a b l y this is due to a sensitivity of D N A synthesis to dilution rather t h a n to varying the ratio of extract to template D N A as shown b y the following experiments: A 5-fold dilution of an extract from TTs, ~infected cells b y increasing the volume of the reaction mixture resulted in a

80

E. Scherzinger and F. Litfin T73,6 &O0o

E 3002

E 2000I00-o o T73,&,6

o I~0

115

2'0

Celt extract added ( p[] Fig. 4. Effect of cell extract concentration on ¢X174 DNA-directed DNA synthesis. DNA synthesis was measured in the standard assay (50 ~zl), except that the extract concentration was varied as indicated. The final KC1 concentration in all reactions was 80 raM. The cell extracts were prepared from E. coli H559 infected with the indicated T7-mutants as described under "Methods ". Incubation was performed at 30 ° C for 4 rain

Table 3. Effect of extract dilution on the rate of DNA synthesis Reaction volume (~1)

Activity

~:a,6

T7a,4,6 %

50 250

too (ll2) it (52)

s (76) 5 (42)

Reaction mixtures (final volume either 50 ,~l, or 250 ~I) contained 0.25 ~g ~)X174 DNA and 10 ~l of an extract prepared from cells infected with the indicated T7-mutants. The concentrations of all the others components were those listed in the standard assay (see "Methods"). 100% activity correspond to 85 pmolcs dTM~ incoporated in 2.5 rain at 30°C. Values in parentheses were obtained using about 0.25 Fg RNA-primed ~)X174 DNA as template-primer.

n e a r l y 10-fold r e d u c t i o n in t h e r a t e of D N A s y n t h e s i s (Table 3). This s t r i k i n g effect was n o t o b s e r v e d w h e n R N A - p r i m c d ¢bX174 D N A was u s e d as t e m p l a t e , i n d i c a t i n g t h a t t h e dilution-sensitive step in t h e r e a c t i o n was D N A s t r a n d initiation. As shown in Fig. 5, D N A synthesis was r e l a t i v e l y r e s i s t a n t t o s u b s e q u e n t dilution, it t h e e x t r a c t s h a d been p r e i n c u b a t e d for a s h o r t p e r i o d w i t h t h e t e m p l a t e D N A in t h e presence of ribonucleoside t r i p h o s p h a t e s . Omission of t h e r N T P s from t h e p r e i n e u b a t i o n m i x t u r e r e s u l t e d in a 5-fold r e d u c t i o n in t h e initial r a t e of D N A synthesis. F r o m these findings we p r e s u m e t h a t for efficient D~qA chain initiation, in a d d i t i o n to ribonueleoside t r i p h o s p h a t e s , t h e presence of one or more e x t r a c t c o m p o n e n t s a t high c o n c e n t r a t i o n is needed. On t h e o t h e r h a n d , w i t h 10 ~l of a n e x t r a c t p r e p a r e d f r o m f r o m T7a,6-infected cells, t h e r a t e of D N A s y n t h e s i s r e m a i n e d essentially c o n s t a n t over a wide range

F u n c t i o n of Phage T7 Gene 4 Product

I ~

81

Preincubation y+rNTP

k

1

I

I

3 Time 30°C(min) 2

I

4

Fig. 5. Dilution-resistant D N A synthesis depends on preincubation with ribonucleoside triphosphates. Reaction mixtures (50 ~zl, without the four dNTPs) containing 3.4 nmoles ~)X174 DNA and l0 ~1 of a n extract from T7s, B-infected cells were preincubated for 2.5 m i n a t 30 ° C in the presence or absence of the four r N T P s as indicated. A t time zero, 200 F1 of a solution containing the components omitted from the preincubation mixture were added. The final concentrations of all reaction components after dilution were those listed in the standard assay (see Methods), except t h a t the extract and the template-DNA were 5-fold diluted. A t the indicated times aliquots were withdrawn from the reaction mixture and assayed for [sH] dTMP incorporation

2oo!

____._._.___..~--~,L-r T73,6

.~ 150"

~ 100"50a. i--

>.....~.. ~ - - ~ ~

1

~

~.

"[ 73/.,6

~

~

I,,17

¢~X174DNA/reaction mixture {nmoles) Fig. 6. Effect of template DNA concentration on the rate of DNA synthesis. S t a n d a r d reaction mixtures were used, except t h a t the assay mixtures contained the indicated amounts of ¢X174 DNA. Incubation was for 4 rain a t 30 ° C with 10 F1 of extracts prepared from T7~.~- or T73.4.6-infected cells

of DNA concentration (Fig. 6). Considering that 10 F1 of extract contained protein of ~-~7 × l 0 s T7-infected cells, this range represents about 150 to 3.000 (~X174 DNA molecules per equivalent of infected cell.

Role o] T7 R N A Polymerase In a previous study (Seherzinger and Litfin, 1974) we have reported that a combination of T7 RNA polymerase, T7 DNA polymerase and E. coli unwinding 6a

~Iolec. gen. Genet. 135

82

E. Scherzinger and F. Litfin

Table 4. T7 RNA and DNA polymcrase activity in extracts from T7-infected E.coli H559 Cell Extract

T7a ~ (15 rain a) T73'~~ (15 rain) T7aisi~ (15 rain)

Activity T7 RNA polymerase %

T7 DNA polymerase %

100 102 126

100 93 <2

The assay conditions were as described under "Methods", using 0.5 ~1 of extracts prepared from cells infected with the indicated T7 mutants. Nucleotide incorporation was linear in the range from 0,25 to 1 ~l of cell extract used. 100% RNA polymerase activity was equivalent to 190 pmoles rCMP incorporated in 10 rain at 30° C; 100% DNA polymerase activity was equivalent to the incorporation o~ 85 pmoles dTMP. a Time given is post-infection.

protein (Sigal e~ al., 1972) was sufficient to replicate ~X174 DNA in vitro. In this model system T7 RNA polymerase synthesizes an R N A primer which is subsequently extended by T7 DNA polymerase. The role of the E. cell unwinding protein in the reaction is twofold: I t limits the primer synthesis by masking the template DNA and it stimulates T7 DNA polymerase in the extension of the R N A primer fragment. Similar effects were observed when the E. cell unwinding protein was replaced b y the T7-induced DNA-binding protein (Scherzinger et al., 1973); in addition, this system can now accept native T7 DS~A as template (unpublished experiments). The DNA synthesizing activity observed in crude extracts from T7-infected cells, however, appears to be different from the reaction catalyzed b y the purified enzymes. As shown in Table 4, extracts from both T7a, 6- and T7a,~,~-infeeted cells contained comparable levels of T7 R N A polymerase and DNA polymerase activity, when tested with native T7 D N A or denatured calf thymus DNA, respectively. Both T7 mutants induced also normal amounts of the DNA-binding protein as judged by analysis of the protein patterns given by each T7 m u t a n t on SDS-polyacrylamide gels (according to Studier, 1972). Nevertheless, efficient conversion of ~bX174 DNA to its replicative form in crude extracts from T7infected cells required the presence of the gene 4 product. Attempts to measure ~X174 DNA-directed RNA primer synthesis in extracts from T7-infccted cells were as yet unsuccessful. As seen in Fig. 7, the action of purified T7 R N A polymerase on ~X174 DNA was strongly inhibited b y addition of increasing amounts of extracts prepared from both T73, ~- and TTs,~,6-infected cells. With extracts at optimal concentration for DNA synthesis no significant rifampicin-resistant R N A primer synthesis was measurable, even though the extracts have been supplemented with exogenous T7 R N A polymerase. Thus, our experiments do not clearly indicate an R N A synthetic event. Accurate determinations, however, have been hampered by ~ low background of unspecific I%NA synthesis which was independent of added ~bXi74 DNA.

Function of Phage T7 Gene 4 Product

83

% 100~

E

>

50-

P_ ,

~

,

2.5 5 7.5 Cell extract added (p[)

r

10

II~-~ --

20

Fig. 7. Inhibition of T7 RNA polymerase activity on ¢X174 DNA by extracts from T7infected E. coli H559. RNA synthesis was measured in standard reaction mixtures (without the four dNTPs) containing 0.25 mM each of the four rNTPs (V3H]rCTP), purified T7 RNA polymerase and various amounts of extracts prepared from cells infected with the indicated T7-mutants. Without added cell extract, T7 RNA polymerase catalyzed the incorporation of 800 pmoles rCMP in 10 min at 30° C

Complementation in Extracts from T7-infected Cells Extracts lacking either the T7 gene 4 or gene 5 product could stimulate DNA synthesis in the opposite extract only with T7 DkTA but not with ~)X174 DNA as template. This is demonstrated by the following experiments (Fig. 8A and B): The reactions shown in Fig. 8A contained native T7 DNA as template, l0 ~zl of a receptor extract prepared from T73,4,e-infccted cells and increasing amounts of extracts to be tested for complementation. Addition of the same extract led only to a slight increase in DNA synthesis. Although not shown in Fig. 8, an extr~ct from T73,5,e-infccted cells by itself had little if any DNA synthesizing activity. However, the activity found in a mixed extract was more than additive, resulting in about 50% restoration of the activity obtained with 10 F1 of an extract from T73,e-infected cells. B y contrast, using ~X174 DNA as template the gene 4 and gene 5 products could not complement at all in vitro (Fig. 8B). When different amounts of extracts from T7~,e-infected cells were mixed with 10 F1 of a gene 4- extract, the resulting activity corresponded nearly to that obtained with the T73,e-extraet by itself. Similar results have been obtained using receptor extracts deficient in the gene 5 product (not shown).

Discussion

T7 D57A is replicated in vivo by a discontinuous mechanism involving the synthesis of small fragments (Masamune et al., 1971). The initiation of "Okazaki fragments" is a process of particular interest, since none of the known DNA polymerases is capable of starting a DNA chain de novo. By whatever mechanism,

84

E. Scherzinger and F. Litfin

(A)

T73'5'6

(s)

80-6

600-

T%G

~ 60-

/

/

a

./

z.0040-

/

//

./

200-

~ 20"0

....... 4~.//

O.

0.50

0.75 1.00 Ratio of extracts

01~

T73,5,6. I

050

I

0.75

T7346 I

1.00

(V/V)

Fig. 8A and B. Complementation in extracts from T7-infected cells. The receptor extract (10 tzl per assay) was prepared from cells infected with T73. a.6 as described under "Methods ". In addition, each reaction mixture contained the indicated amounts of extracts to be assayed for complementing activity (final volume of the mixed extracts: 20 ~l). (A) The reaction mixtures (0.1 ml) contained the components and concentrations of the standard assay with the following exceptions: ¢X174 DNA was replaced by T7 DNA (1.3 Fg). MgCI~ concentration was 20 mM and the four 4NTPs were present at 0.1 mM each. Incubation was for 20 min at 30 ° C. (B) ¢X174 DNA-directed DNA synthesis was measured in 50 F1 standard reaction mixtures after incubation for 10 min at 30 ° C. The final KCI concentration was 80 mM

in T7 r e p l i c a t i o n t h e p r o d u c t of gene 4 a p p e a r s in s a m e w a y to be i n v o l v e d in t h e g e n e r a t i o n of t h e D N A f r a g m e n t s (Wolfson a n d Dressier, 1972). I n o r d e r to e l u c i d a t e t h e f u n c t i o n of this protein, t h e r e p l i c a t i o n of a n u n p r i m e d s i n g l e - s t r a n d e d D N A (~bX174 D N A ) b y a soluble e n z y m e f r a c t i o n e x t r a c t e d from T7-infected cells was studied. A l t h o u g h this in vitro s y s t e m does n o t s i m u l a t e t h e n a t u r a l conditions, it p r o v e d to be useful i n a s m u c h as t e m p l a t e c o p y i n g was d e p e n d e n t on active T7 gene 4 a n d gene 5 p r o d u c t s . B o t h of these gene p r o d u c t s are k n o w n to be r e q u i r e d for T7 D N A r e p l i c a t i o n in rive. Our studies of this s y s t e m n o w d e m o n s t r a t e t h a t t h e gene 4 p r o d u c t functions in c o m p l e m e n t a r y s t r a n d i n i t i a t i o n b y a m e c h a n i s m which does n o t involve a c o v a l e n t linkage of t h e p r o d u c t to t h e t e m p l a t e D N A . The gene 4 p r o d u c t is dispensable for i n i t i a t i o n of D N A synthesis on R N A - p r i m e d ~bX174 D N A , suggesting t h a t its a c t i o n is n o t p r i m a r i l y due to assisting T7 DxNA p o l y m e r a s e in a m o r e effective u t i l i z a t i o n of p r e - e x i s t i n g 3 ' - 0 t t t e r m i n a t e d p r i m e r chains. Efficient D N A s t r a n d i n i t i a t i o n in e x t r a c t s from T7-infccted cells exhibits n o t only a n e a r l y a b s o l u t e r e q u i r e m e n t for t h e p r o d u c t s of T7 genes 4 a n d 5, b u t is also d e p e n d e n t u p o n a d d e d ribonucleoside t r i p h o s p h a t c s . The l a t t e r requirem e n t p o s s i b l y p o i n t s to a p a r t i c i p a t i o n of T7 R N A p o l y m c r a s e in t h e i n i t i a t i o n reaction. However, t h e fact t h a t s u b s t a n t i a l D N A s t r a n d i n i t i a t i o n occurs w i t h each one of t h e single ribonucleoside t r i p h o s p h a t e s argues a g a i n s t a n o r m a l R N A s y n t h e t i c e v e n t a n d suggests i n s t e a d t h a t some novel m e c h a n i s m m a y f u n c t i o n in D N A chain initiation. A r e m a r k a b l e f e a t u r e of t h e T7 in vitro r e p l i c a t i o n s y s t e m is t h a t gene 5 e x t r a c t s can c o m p l e m e n t gene 4 - e x t r a c t s w i t h n a t i v e T7 D N A b u t n o t

Function of Phage T7 Gene 4 Product

85

with ~X174 D N A as template. This observation suggests t h a t the gene 4 and gene 5 products are parts of a larger structure formed in vivo, the formation of which is mediated in vitro only b y th e proper double-stranded DNA. Although double strand-like regions are k n o w n to exist also in djX174, M13 and fd D N A (Schaller et al., 1969; Forsheit and R a y , 1970), preferential binding of the separate components to extended single-stranded DN~A regions m a y prevent their appropriate arrangement to a functional starting complex. A cooperative interaction of the various components of the system for starting replication is also indicated b y the unusual sensitivity of the initiation complex to dilution. A precise characterization of the role of the T7 gene 4 p r o d u c t in D N A chain initiation m u s t await further purification of all the components involved in this reaction. B y use of DNA-cellulose chromatography, we have isolated the gene 4 p r o d u c t to a b o u t 80 % p u r i t y (unpublished results). A double-labeling technique confirmed the identity of the isolated protein. I t has a molecular weight of 65000 daltons as judged b y SDS-gel electrophoresis. The purified protein binds weakly and preferentially to single-stranded DNA, b u t its enzymatic function is as y e t unknown.

Acknowledgement. We would like to thank H. Schuster for his generous support and helpful advice in preparation of the manuscript. References Biicher, T.: t~ber ein phosphatfibertragendes G~rungsferment. Biochim. biophys. Acta (Amst.) l, 292 (1947) Burgess, R. R.: A new method for the large scale purification of E. coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J. biol. Chem. 244, 6160 (1969) Center, M. S., Richardson, C. C.: An endonuclease induced after infection of E. coli with bacteriophage T7. J. biol. Chem. 245, 6285 (1970) Forsheit, A. B., Ray, D. S. : Conformations of the single-stranded DNA of bacteriophage M13. Proc. nat. Acad. Sci. (Wash.) 67, 1534 (1970) Francke, B., Ray, D. S. : Fate of parental tX174-D~N'A upon infection of starved thyminerequiring host cells. Virology 44, 168 (1971) Grippo, P., Richardson, C. C. : Deoxyribonucleic acid polymerase of bacteriophage TT. J. biol. Chem. 246, 6867 (1971) Hinkle, D. C., Richardson, C. C. : Bacteriophage T7 deoxyribonucleic acid replication in vitro. J. biol. Chem. 249, 2974 (1974) Masamune, ¥., Frenkel, G. D., Richardson, C. C.: A mutant of bacteriophage T7 deficient in polynucleotide ligase. J. biol. Chem. 246, 6874 (1971) Oey, J. L., Str~tling, W., Knippers, R. : A DNA polymerase induced by bacteriophage T7. Europ. J. Bioehem. 23, 497 (1971) Schaller, H., Voss, H., Glucker, S. : Structure of the DNA of bacteriophage fd. J. molec. Biol. 44, 445 (1969) Schekman, R., Wiekner, W., Westergaard, O., Brutlag, D., Geider, K., Bertsch, L. L., Kornberg, A. : Initiation of DNA synthesis: synthesis of 6X174 replicative form requires RNA synthesis resistant to rifampiein. Proc. nat. Acad. Sci. (Wash.) 69, 2691 (1972) Scherzinger, E., Litfin, F. : Initiation of the replication of single-stranded DNA by concerted action of phage T7 RNA and DNA polymerases. Europ. J. Biochem. 46, 179 (1974) Scherzinger, E., Litfin,F., Jost, E.: Stimulation of T7 DN'A polymerase by a new phagecoded protein. Molec. gen. Genet. 128, 247 (1973) Sigal, N., Delius, H., Kornberg, T., Gefter, M. L., Alberts, B. ]~I.: A DNA-unwinding protein isolated from E. coli: its interaction with DNA and with DNA polymerases. Proc. nat. Acad. Sci. (Wash.) 69, 3537 (1972) 6b ~olec. gen. Genet. 135

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Sinsheimer, R. L. : ¢X174 DNA. Proc. Nucl. Acid Res. 1, 569 (1966) Sobell, H.M., Jain, S.C., Sakore, T.D.: Stereochemistry of aetinomycin-DI~A binding. Nature (Load.) 231, 200 (1971) Str~tling, W., Ferdinand, F. J., Krause, E., Knippers, R.: Bacteriophage T7 DNA replication in vitro: an experimental system. Europ. J. Biochem. 38, 160 {1973) Str~tling, W., Knippers, R. : Function and purification of gene 4 protein of phage T7. Nature (Load.) 245, 195 (1973) Studier, F. W.: The genetics and physiology of bacteriophage T7. Virology 39, 562 (1969) Studier, F. W. : Bacteriophage T7. Science 176, 367 (1972) Tabak, H . F . , Griffith, J., Geider, K., Schaller, H., Kornberg, A.: Initiation of deoxyribonucleic acid synthesis. J. biol. Chem. 249, 3049 (1974) Thomas, C. A., Jr., Abelson, J. : The isolation and characterization of DNA from bacteriophage. Proe. Nucl. Acid Res. 1, 553 (1966) Wickner, W., Schekman, R., Geider, K., Kornberg, A. : A new form of DIqA polymerase I H and a copolymerase replicate a long, single-stranded primer-template. Proc. nat. Acad. Sci. (Wash.) 79, 1764 (1973) Wolfson, J., Dressier, D. : Regions of single-stranded DNA in the growing points of replicating bacteriophage T7 chromosomes. Proc. nat. Acad. Sci. (Wash.) G9, 2682 (1972) Communicated by W. Arber Eberhard Seherzinger Frank Litfin Max-Planck-Institut ffir Molekulare Genetik Abteilung Schuster Ihnestral~e 63-73 D-1000 Berlin 33