BIOCHIMIE, 1976, 58, 1101-1111.

In vitro synthesis of simian virus 40 DNA. I. - Synthesi~ b y a soluble extract from infected CV1 cells. M a r c GIRARD

~, L o u i s e MART¥,C h a n t a l CAJEAN a n d F r a n c o i s e SUAREZ.

Unitd de Physiologie des Virus, I n s t i t u t d e R e c h e r c h e s S c i e n t i f i q u e s s u r le C a n c e r , C.N.R.S., B.P. n ° 8, 94800 V i l l e j u i f ( F r a n c e ) . (15-6-1976).

S u m m a r y . - - S i m i a n Virus 40 (SV4O) DNA replication was studied in vitro using cell free extracls p r e p a r e d f r o m SV40 infected CV1 cells. The cells were f r a c t i o n a t e d into a soluble cytoplasmic fraction a n d nuclei. The nuclei were lysed 'with h i g h salt a n d used to prepare a soluble nuclear fraction. Both f r a c t i o n s displayed DNA polymerase activity as m e a s u r e d w i t h activated calf t h y m u s DNA. However, only the cytoplasmic f r a c t i o n was active whe.n SV40 DNA c o m p o n e n t I molecules were used as template. Under these conditions, the cytoplasmic e x t r a c t was s h o w n to catalyse the SV4O DNA dependent, in vitro i n c o r p o r a t i o n of the four deoxyribonucleotides into DNA molecules w h i c h had, at b o t h n e u t r a l ,and .alkaline pH, the sam.e s e d i m e n t a t i o n b e h a v i o r as authenti,c SV40 DNA compon e n t I a n d componen.t II molecules. Optimal Mg ++ c o n c e n t r a t i o n was 5-8 inM. I n c o r p o r a t i o n of label into DNA c o m p o n e n t I molecules showed a n i n i t i a l lag of a b o u t 15 min., a f t e r w h i c h it vcas l i n e a r w i t h t i m e for up to 5 hrs at 32 °. I n c o r p o r a t i o n into DNA eompon.ent II molecules proceeded w i t h o u t obvious lag a n d reached a plateau a f t e r a p p r o x i m a t e l y 2 h r s of i n c u b a t i o n . It is concluded t h a t the eytopl.asmic exlract supports the in vitro synthesis of SV40 DNA a n d t h a t DNA c o m p o n e n t II molecules a p p e a r to be a precursor to DNA c o m p o n e n t I molecules in t h e reaction. Labeling of viral DNA molecules was highly dependent on ATP a n d on a n ATP generating system. In the absence of ATP and of the energy generating system, i n c o r p o r a t i o n occurred but b o t h t e m p l a t e and newly synthesized DNA Inol~eeules w.ere extensively degraded.

INTRODUCTION. S e v e r a l cell f r e e s y s t e m s h a v e b e e n d e s c r i b e d f o r t h e s t u d y of DN~A r e p l i c a t i o n i n b o t h u n i n f e c t e d a n d v i r u s i n f e c t e d m a m m a l i a n c e l l s [1, 5, 6, 8, 10, 13, 16, 17, 19, 21, 24, 34]. T h e s e s y s t e m s i n v o l v e t h e u s e of e i t h e r c r u d e c e l l l y s a t e s o r i s o l a t e d nuclei. Although much has been learnt from them regard i n g t h e e l o n g a t i o n a n d t e r m i n a t i o n of D N A s y n t h e s i s , t h e y h a v e n o t a l l o w e d so f a r t h e c h a r a c t e r i z a t i o n a n d p u r i f i c a t i o n of D N A r e p l i c a t i o n fact o r s i n a n i m a l cells. O n e r e a s o n is t h a t t h e y a r e n o t a b l e to s u p p o r t t h e d e n o v o i n i t i a t i o n of D N A s y n t h e s i s ( a l t h o u g h i n i t i a t i o n of O k a s a k i t y p e f r a g ments occurs) but can only elongate nascent DNA s t r a n d s a l r e a d y i n i t i a t e d i n t h e cell, p r i o r t o t h e p r e p a r a t i o n of t h e l y s a t e . A n o t h e r r e a s o n w h y t h e s e c r u d e s y s t e m s a r e n o t q u i t e s u i t a b l e is t h a t r e p l i c a t i o n of v i r a l D N A ' s s u c h as t h a t of SV40 o r p o l y o m a v i r u s is o b s c u r e d b y t h e c o n t i n u e d o c c u r r e n c e of h o s t cell D N A s y n t h e s i s a n d / o r r e p a i r . L a s t , b u t n o t least, t h e s e c r u d e s y s t e m s d o To w h o m all correspondence should be addressed.

not provide the investigator with the possibility of c o n t r o l l i n g t h e n a t u r e , t h e s t a t e , o r t h e p h y s i c a l e n v i r o n m e n t of t h e D N A t e m p l a t e . It is h o w e v e r o b v i o u s t h a t SV40 a n d p o l y o m a virus could be ideal probes for the in vitro study of D N A r e p l i c a t i o n i n m a m m a l i a n cells, s i n c e t h e i r m e c h a n i s m of r e p l i c a t i o n is w e l l k n o w n f r o m in vivo studies, and since elongation and termination of t h e i r r e p l i c a t i o n a p p e a r to b e e n t i r e l y c a r r i e d o u t b y c e l l u l a r g e n e p r o d u c t s . T h e e x i s t e n c e of t h e ts A m u t a n t s of b o t h t h e s e v i r u s e s [7, 9, 29, 30], i n w h i c h i n i t i a t i o n of D N A s y n t h e s i s is b l o c k e d i n v i v o , p r o v i d e s a u n i q u e m e a n s of s t u d y i n g t h e i n i t i a t i o n of D N A r e p l i c a t i o n i n a n a n i m a l cell s y s t e m . Also, t h e s t u d y of SV40 o r p o l y o m a D N A r e p l i c a t i o n in v i t r o m i g h t s h e d s o m e l i g h t o n t h e b a s i c p r o b l e m of w h y a c e l l is o r n o t p e r m i s s i v e to t h e r e p l i c a t i o n of a g i v e n v i r a l g e n o m e . T h e s e c o n s i d e r a t i o n s p r o m p t e d u s to p r e p a r e a s o l u b l e , cell f r e e , D N A f r e e a n d D N A d e p e n d e n t , e x t r a c t f r o m SV40 i n f e c t e d cells, r a t h e r t h a n a cell f r e e l y s a t e . W e s h a l l d e s c r i b e a s y s t e m w h i c h is a b l e , w h e n s u p p l e m e n t e d w i t h SV40 DNA, t o i n c o r porate the four deoxyribonucleotides linearly with

M. Girard a n d coll.

1102

time, d u r i n g at least 5 hrs, into a m a t e r i a l exhibiting the s e d i m e n t a t i o n properties of a u t h e n t i c SV40 DNA. MATERIAL AND METHODS.

Cells and Viruses. S u b c l o n e d CV 1 cell [22] were seeded i n glass Roux bottles at a density of 5 X 106 cells per bottle a n d i n c u b a t e d at 37°C u n d e r growth m e d i u m (MEM s u p p l e m e n t e d w i t h 10 per cent tryptose phosphate, 5,0,0 U / n i l of p e n i c i l l i n G, 100 ,~g/ml of s t r e p t o m y c i n sulfate, and 3.18 g glucose per liter added -with 1,0 per cent calf serum). The m e d i u m was c h a n g e d on day 3. On day 4, the cells were infected w i t h w t SV4~0 [29], at a m u l t i p l i c i t y of a p p r o x i m a t e l y 5 pfu/cell. Adsorption was for 2 hrs at room t e m p e r a t u r e , after w h i c h the cell culture was i n c u b a t e d for 65-70 hrs at 3~3°C u n d e r growth m e d i u m added with 2 per cent calf serum. I n most experiments, the cultures were t h e n t r a n s f e r r e d for 2 hrs to 41°C p r i o r to collection of the cells. This p r o c e d u r e regularly y i e l d e d about 5 X 107 infected cells per Roux bottle. T h e w t s t r a i n of SV40 was a gift from P. Tegtmeyer. Stocks of v i r u s w e r e p r e p a r e d u s i n g plaque purified v i r u s a n d i n f e c t i n g the cells at an i n p u t m u l t i p l i c i t y of a p p r o x i m a t e l y 10 -4 pfu/cell. Stocks were collected after 19-21 days at 33°C.

Preparation o[ DNA. SV40 DN,A c o m p o n e n t I was p r e p a r e d a c c o r d i n g to Hirt [11] from CV1 cells g r o w n i n 10 cm Petri dishes a n d infected 13 days p r e v i o u s l y w i t h wt SV46, at an i n p u t m u l t i p l i c i t y of a p p r o x i m a t e l y 10.-4 pfu/cell. The low M.W. DNA o b t a i n e d from the Hirt e x t r a c t i o n s u p e r n a t a n t was d e p r o t e i n i z e d 3 times w i t h p h e n o l a n d c h l o r o f o r m (1:1~), precipitated w i t h 2 vol. of 95 per cent ethanol at - - 2 0 ° C , t h e n r e s u s p e n d e d into 10-2M Tris-HC1 pH 7.4, 0.1 × SSC. The DNA was added w i t h 100 ~ g / m l e t h i d i u m b r o m i d e a n d adjusted to a final density of 1.58 g/cm3 w i t h solid CsCl. The m i x t u r e was c e n t r i f u g e d for 40 hrs at 40,000 rpm, 150C, i n the 50 t i t a t i u m rotor of the Spinco. After centrifugation, the tubes were p i e r c e d a n d fractions collected dropwise. Those c o n t a i n i n g the b a n d of s u p e r h e l i c o i d a l DNA (DNA c o m p o n e n t I) were centrifuged again to e q u i l i b r i u m t h r o u g h a n e w CsC1 g r a d i e n t i n the p r e s e n c e of e t h i d i u m bromide. The r e s u l t i n g b a n d of twice purified DNA c o m p o n e n t I was extracted 3 times w i t h 1 volume isopropanol, dialyzed for a p p r o x i m a t e l y

BIOCHIMIE, 1 9 7 6 ,

58, n ° 9.

16 hrs against a large volume of 10 -2 M Tris-HC1 pH 7.4, 10 -4 M EDTA (TE buffer) a n d stored frozen at a c o n c e n t r a t i o n of 500 i~g/ml. Labeled SV40 DNA molecules w e r e p r e p a r e d similarly, except that the cell cultures w e r e labeled w i t h the r e q u i r e d radioactive p r e c u r s o r ([aH~ t h y m i d i n e or 32p, C.E.A., F r a n c e ) for 2 days before the e x t r a c t i o n of the DNA. Labeling w i t h 30 ~ C i / m l 32p for that length of time t y p i c a l l y y i e l d e d v i r a l DNA "~vith a specific activity of a p p r o x i m a t e l y 3 × 104 cpm/.~g. Activated calf t h y m u s DNA (Boehringer) was p r e p a r e d a c c o r d i n g to W i c k n e r et al [33] a n d stored frozen at a c o n c e n t r a t i o n of 2 m g / m l .

Preparation of extracts. Crude c y t o p l a s m i c extracts were p r e p a r e d after Kidwell a n d Mueller [171, from 2,0 Roux bottles. The cells were s c r a p e d i n w a r m phosphate buffered saline (PBS) w i t h the aid of a silicone r u b b e r , c e n t r i f u g e d at room t e m p e r a t u r e , resusp e n d e d i n w a r m PBS, a n d s e d i m e n t e d again. The pellet was r e s u s p e n d e d into 4 volumes of ice cold h y p o t o n i c buffer (buffer A : 10 mM potassium phosphate pH 7.8, 3 mM MgC12, 1raM EDTA, 2 mM dithioerythritol). The cells w e r e allowed to swell for 10 min. at + 4°C t h e n lysed by seven strokes of a tight fitting Dounce homogenizer (Kontes Glass Co). I m m e d i a t e l y after lysis, i s o t o n i c i t y was restored to the cell extract t h r o u g h the a d d i t i o n of o n e - t h i r d volume of buffer B (240 mM Tris-HG1 pH 8.'0, 100 mM glucose, 4'00 mM NaC1, 1 mM EDTA, 2 mM dithioerythritol). Nuclei were t h e n s e d i m e n t e d by c e n t r i f u g a t i o n for 10 min. at 650 g, + 2°C. The resulting s u p e r n a t a n t (cytop l a s m i c extract) was ikept i n ice w h i l e the pellet (nuclei) was resuspellded into one half to one t h i r d the original volume of buffer A, d o u n c e d again, a d d e d w i t h 1/3 volume of buffer B, a n d s e d i m e n t e d for 10 min. at 2.00'0 g t h r o u g h a 20 per cent w / w sucrose c u s h i o n i n buffer C (see below). This second c e n t r i f u g a t i o n step resulted in the f r a c t i o n a t i o n of the crude n u c l e a r homogenate into three l a y e r s : a soluble c y t o p l a s m i c fraction at the top, a p a r t i c u l a t e f r a c t i o n (cell debris a n d u n b r o k e n cells) at the surface of the sucrose cushion, a n d a pellet (nuclei) at the bottom of the tube. The u p p e r f r a c t i o n was carefully r e m o v e d a n d a d d e d to the p r e c e d i n g c y t o p l a s m i c extract. The cell debris were r e s u s p e n d e d into a small volume of buffer A, homogenized again w i t h the aid of the Dounce homogenizer, a n d sedim e n t e d at 2 000 g t h r o u g h a n e w 20 per cent sucrose cushion. The cytoplasmic f r a c t i o n from

S V 4 0 DNA s y n t h e s i s i n vitro. this last c e n t r i f u g a t i o n was added to the p r e c e d i n g ones, a n d the n u c l e i were pooled w i t h those from the p r e c e d i n g centrifugation. The pooled soluble fractions r e p r e s e n t i n g the cytoplasmic extract w e r e s e d i m e n t e d for 9.0 rain. at 100,000 g, 2°C, i n the 50 t i t a n i m n rotor of the Spinco. The resulting s u p e r n a t a n t ($10~)) was c o n c e n t r a t e d t h r o u g h p r e c i p i t a t i o n by 80 per cent saturated a m m o n i u m sulfate in the cold. The extract was stored u n d e r the form of an ammon i u m sulfate pellet at - - 8 0 ° C . The pooled nuclei were r e s u s p e n d e d in a small volume of buffer C (60 mM Tris-HG1 pH 8:0, 5 mM potassium phosphate pH 7.8, 3 mM MgC1.2, 2 mM d i t h i o e r y t h r i t o l , 1 mM EDTA, 25 mM glucose, 1,00 mM NaC1) a n d treated w i t h 0.25 per cent T r i t o n X 10,0. T h e y were t h e n w a s h e d w i t h buffer C after w h i c h they were extracted w i t h 0.2 M potassium phosphate, pH 7.8 [28] i n the p r e s e n c e of 2 M KC1, at a c o n c e n t r a t i o n of about 2 X 10s nuclei p e r ml. The resulting viscous lysate was clarified b y c e n t r i f u g a t i o n for 90 rain at 100,000 g, 2°C, i n the S W 5 0 rotor of the Spinco. The s u p e r n a t a n t , r e p r e s e n t i n g the n u c l e a r extract (N 1'00) ~ a s diluted 3 fold w i t h buffer C, t h e n precipitated w i t h a m m o n i u m sulfate and stored as described for the cytoplasmic extract.

Standard conditions for in vitro DNA synthesis. T h e n u c l e a r (N1,00) or cytoplasmic (S100) a m m o n i u m sulfate precipitates were dialyzed against 25 mM Tris-HCl pH 7.5, 60 mM KC1, 5 mM ~-mercaptoethanol, 2 mM EDTA, and 10 per cent glycerol (sample buffer) and in most e x p e r i m e n t s adjusted to 5 m g / m l p r o t e i n w ' t h sample buffer. The dialyzed extracts could be stored at - - 8 0 ° for a couple of weeks w i t h o u t a p p a r e n t loss of activity. S t a n d a r d assays for in vitro SV40 DNA synthesis were p e r f o r m e d at 32°C in a final volume of 0.1 ml c o n t a i n i n g 4'0-5.0 ~g/ml SV4'0 DNA comp o n e n t I. C o n c e n t r a t i o n of the assay m i x t u r e was 20 mM Tris-HC1 pH 7.7 (or 40 mM Hepes pH 7.8) 6 mM MgCle, 5 mM d i t h i o e r y t h r i t o l , 100 ,~M each of d A T P and dCTP, 50, :~M each of dGTP and dTTP, 1 mM ATP, 6 mM p h o s p h o e n o l p y r u v a t e (PEP), 5 :~g/ml p y r u v a t e kinase, 26'0, ,~g/ml E. colt tRNA (Boehringer), T.5 mM p u t r e s c i n e and 30 mM KCI. In addition, the extracts (.,0.25 ml) c o n t r i b u t e d 6.25 mM Tris pH 7.5, 1!5 mM KC1, 1.25 mM ,~ mercaptoethanol, 0.5 mM EDTA, and 2.5 per cent glycerol. SV40 DNA c o m p o n e n t l stored in TE buffer at - - 8 0 ° C was diluted to 2'00 u g / m l w i t h l:0-e M Tris-HC1 pH 7.4, 5 mM MgC12, p r i o r to use. It

BIOCHIMIE, 1976, 58, n ° 9.

1103

therefore c o n t r i b u t e d a p p r o x i m a t e l y 0.5 mM Mg÷" and 1 mM Tris to the final c o n c e n t r a t i o n s . Labeling of the p r o d u c t of the reaction was t h r o u g h the use of [3HI d T T P a n d / o r [3H~ dGTP (New E n g l a n d Nuclear) at the i n d i c a t e d specific activities. Bul:k DNA polymerase activity was assayed in the presence of 2,0 r~g activated calf t h y m u s DNA at 37°C in a final volume of 0.1 ml. Assay m i x t u r e was 10 mM MgC12, 5 mM dithioerythritol, 20 mM Tris-HC1 pH 7.7 or 8.8, w i t h 100 '!~M each of dATP, dCTP and dGTP, 50 ~M of [3HI dTTP at the indicated specific activity, and 20 !~g b o v i n e serum a l b u m i n e (BSA). One u n i t of DNA polymerase activity was defined as the a m o u n t of p r o t e i n that i n c o r p o r a t e s 1 nmole of dTMP into DNA in 6.0 rain. at the i n d i c a t e d temperature. I n c o r p o r a t i o n of radioactive p r e c u r s o r s was stopped by the a d d i t i o n of 0.2 ml ice cold 0.2 M Na p y r o p h o s p h a t e c o n t a i n i n g 10.0 .ug/ml bovine s e r u m a l b u m i n (BSA), followed by 5 ml of ice cold 5 per cent t r i c h l o r a c e t i c acid (TCA). The resulting precipitates were collected on GF/C glass fiber filters (Whatman), r i n s e d w i t h approximately 20 ml 1 per cent TCA followed by 5 ml absolute ethanol, t h e n dried u n d e r i n f r a r e d light a n d c o u n t e d in a toluene based s c i n t i l l a t i o n fluid.

Analysis of the in vitro labeled DNA. For the analysis of in vitro labeled products, i n c o r p o r a t i o n s were stopped by p l a c i n g the tubes in ice and m a k i n g t h e i r content 50 lnM with respect to EDTA. In nmst experiments, SDS was then added to a final c o n c e n t r a t i o n of 0.3 per cent and the m a t e r i a l was analyzed by alkaline sucrose gradient c e n t r i f u g a t i o n t h r o u g h 11 ml 5-20 per cent gradients in 0.3 M NaOH, 0.7 M NaCI, 0:002 M EDTA. Centrifugation was for 12 hrs at 28.00,0 r p m 4°C, in the SW41 rotor of the Spinco. At the end of the c e n t r i f u g a t i o n time, fractions were collected d r o p w i s e from the bottonl of the tubes w i t h the aid of a peristaltic p u m p and their c o n t e n t was processed for d e t e r m i n a t i o n of TCA p r e c i p i t a b l e radioactivity. In other experiments, the i n c u b a t i o n m i x t u r e s were extracted first w i t h phenol, then with p h e n o l and chloroform (1:1) and the DNA was precipitated w i t h ethanol. Part of the DNA was analyzed by n e u t r a l sucrose gradient c e n t r i f u g a t i o n t h r o u g h 11 ml 5-20 per cent sucrose gradients in 0.01 M Tris-HC1 pH 7.4, 1 M NaC1, 0.002 M EDTA. Centrifugation was for 12 hrs at 38,000 rpm, 4°C, in the SW41 rotor of the Spinco. Other portions of the deproteinized DNA sample were analyzed by sucrose gradient c e n t r i f u g a t i o n at alkaline pH

M. G i r a r d a n d coll.

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as d e s c r i b e d above, or by e q u i l i b r i u m c e n t r i f u gation in CsC1 in the p r e s e n c e of 100 !~g/ml ethidium b r o m i d e as d e s c r i b e d for the p r e p a r a t i o n of DNA's. RESULTS. SV40 i n f e c t e d cells w e r e f r a c t i o n a t e d into a n u c l e a r and c y t o p l a s m i c f r a c t i o n as d e s c r i b e d u n d e r Material and Methods, and the 100,I)00, g s u p e r n a t a n t s f r o m each (NI~O9 and Sift0 respectively) w e r e tested for t h e i r in vitro DNA polym e r a s e activity after c o n c e n t r a t i o n by a m m o n i u m sulfate p r e c i p i t a t i o n . T w o tests w e r e c a r r i e d out, w i t h e i t h e r a c t i v a t e d calf t h y m u s DNA or SV4,0 DNA c o m p o n e n t I as template. As w i l l be r e p o r t e d in detail e l s e w h e r e (Cajean, Marry, Suarez and Girard, in p r e p a r a t i o n ) both the S100 and N10,0 f r a c t i o n d i s p l a y e d c o n s i d e r a b l e DNA p o l y m e r a s e a c t i v i t y w h e n assayed w i t h calf t h y m u s DNA, in a g r e e m e n t w i t h the r e p o r t e d dist r i b u t i o n of DNA p o l y m e r a s e s in cell extracts [3, 4, 27, 28, 31, 3~, 35]. H o w e v e r , only t h e S100 fraction displayed noticeable activity when assayed w i t h SV4,0 DNA c o m p o n e n t I molecules. TABLE I.

R e q u i r e m e n t s for SV40 DNA s y n t h e s i s in vitro. SI00 f NI00 (~g proteins)

43

32

Incubation medium complete omit SV40 DNA

Experiment 1 782 72

--

complete omit SV40 DNA

Incorporation (pmoles dTMP) 1.85 •

04

2.98 •

04

1.18

Experiment 2

48

42

omit dNTP omit SV40 DNA omit PEP and PK omit ATP, PEP and PK

1.28 •52 .04 1.72 2.38

Cytoplasmic (S100) and nuclear (N100) fractions were prepared from SV40 infected cells as described under Material and Methods and ineubat,ed for 30 min at 32°C. Complete system was 37 mM Hepes pH 7.8, 1 mM MnC12, 6 mM M,gClo, 2 lnM dithioerythritol, 1 mM ATP, 100 gM each of dATP, dGTP, dCTP, GTP, CTP and UTP, 200 gg/ml of BSA, 40 !~g/ml of SV4~0 DNA component I, 50 ~M[aH]dTTP (260 epm/pmole), and the indicated amount of extract in a final volume of 0.1 ml. The complete system in experiment 2 contained in addition 10 mM phosphoenolpyruvate (PEP) together with 5 ~g/ml pyruvate kinase (P. K.).

BIOCHIMIE, 1976, 58, n ° 9.

This is illustrated in table I, w h i c h shows the result of an assay p e r f o r m e d u n d e r the conditions d e s c r i b e d by H u n t e r and F r a n c k e [13]. Most of the a c t i v i t y m e a s u r e d in the p r e s e n c e of SV4:0 DNA was in the $110,0 extract. This lack of activity in the n u c l e a r extracts w a s not due to the c o n d i t i o n s of the assay, since similar results w e r e o b t a i n e d u n d e r the assay conditions of WinnaCker et al E34] of De P a m p h i l i s and Berg [5] or u n d e r those d e s c r i b e d as s t a n d a r d c o n d i t i o n s u n d e r the section Material and Methods. N e i t h e r w a s it due to the m a n n e r in w h i c h the cells w e r e f r a c t i o n a t e d , since s i m i l a r results w e r e obtained when nuclei were prepared according to S p a d a r i and W e i s s b a c h [28], or w h e n t h e y w e r e p r e p a r e d w i t h o u t d e t e r g e n t (results not shown). Since only the c y t o p l a s m i c S100 f r a c t i o n disp l a y e d substantial a c t i v i t y w i t h SV40 DNA as template, the p r o p e r t i e s of the S10,0 system w e r e i n v e s t i g a t e d further. I n c o r p o r a t i o n w i t h that syst e m w a s fully d e p e n d e n t on t h e a d d i t i o n of SV49 DNA (table I : also, see figure 6 and 7), but only in p a r t on the four d e o x y r i b o n u c l e o s i d e - t r i p h o s phates (table I). T h e fact that the d N T P r e q u i r e m e n t is only p a r t i a l m a y be due to the p r e s e n c e of r e s i d u a l amounts of d N T P ' s in the extract• I n c o r p o r a t i o n w a s .99 p e r cent r e d u c e d ~vhen p a n c r e a t i c DNase (1.0 j.~g/ml) w a s p r e s e n t d u r i n g the assay. P a n c r e a t i c RNase (also 10 igg/ml) h a d no effect. The 3H label was i n c o r p o r a t e d into a m a t e r i a l w h i c h w a s resistant to aIkaline h y d r o l y sis, and resistant to 1'0'0 ,~g/ml RNase (30 rain. at r o o m t e m p e r a t u r e ) . The m a t e r i a l was, on a n o t h e r hand, c o m p l e t e l y digested by DNase (also at 1'09 !~g/ml) in 30 rain. at r o o m t e m p e r a t u r e (not shown). T h e amount of dNMP i n c o r p o r a t e d was p r o p o r t i o n a l to the c o n c e n t r a t i o n of SI0'0 e x t r a c t used, at least in a r a n g e of a p p r o x i m a t e l y 0.1 to 1 m g / m l of protein. H i g h e r c o n c e n t r a t i o n s of e x t r a c t w e r e f o u n d to be i n h i b i t o r y . As s h o w n in figure 1, the system d i s p l a y e d a b r o a d Mg +÷ c o n c e n t r a t i o n o p t i n m m , f r o m a p p r o x i m a t e l y 3 to 8 mM. This w a s true not only of the gross i n c o r p o r a t i o n of d e o x y r i b o n u c l e o t i d e s , as illustrated by figure 1, but also of the l a b e l i n g of DNA m o l e c u l e s w i t h the size and c h a r a c t e r i s t i c s of SV40 DNA c o m p o n e n t I and c o m p o n e n t II. T h e a d d i t i o n of Mn ÷+ to Mg ÷+, or the substitution of Mg ÷÷ by Mn +÷, resulted in a s o m e w h a t v a r i a b l e stim u l a t i o n of dTMP i n c o r p o r a t i o n . It w a s f o u n d h o w e v e r that the p r e s e n c e of Mn ÷÷ d u r i n g incubation could lead to e x t e n s i v e d e g r a d a t i o n of the SV40 DNA template, 'with the e n s u i n g result that most of the label i n c o r p o r a t e d in vitro w a s in short

SV40 DNA synthesis in v i t r o . DNA fragments (not shown). We therefore refrain e d f r o m u s i n g M n ÷÷ i n all t h e l a t t e r e x p e r i m e n t s . T r i s b u f f e r (2,0 mM) c o u l d b e s u c c e s s f u l l y s u b s t i tuted for Hepes buffer, but potassium phosphate b u f f e r , also at 20 raM, w a s i n h i b i t o r y . O p t i m u m p H w a s f o u n d to b e f r o m a p p r o x i m a t e l y 7.5 t o 7.8 (not shown).

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A T P . At t h e e n d of t h e i n c u b a t i o n p e r i o d , t h e s~P l a b e l e d D N A m o l e c u l e s a n d t h e SH l a b e l e d p r o d u c t were analyzed by sucrose gradient centrifugation at e i t h e r a l k a l i n e o r n e u t r a l p H (fig. 2). I n t h e a b s e n c e of A T P ( r i g h t h a n d s i d e p a n e l s i n f i g u r e 2) t h e ~eP l a b e l e d t e m p l a t e D N A m o l e c u l e s w e r e c o n s i d e r a b l y d e g r a d e d as j u d g e d f r o m t h e f a c t t h a t t h e y s e d i m e n t e d as 8-12S m a t e r i a l at n e u t r a l p H ( c l o s e d c i r c l e s , p a n e l B), a n d as 4-8S f r a g m e n t s at a l k a l i n e p H ( c l o s e d c i r c l e s , p a n e l D). S i m i l a r l y , t h e o v e r w h e l m i n g m a j o r i t y of t h e p r o d u c t l a b e l e d

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Fro. 1. - - Influence of the concentration of Mg ++ on the synthesis of SV#O DNA in vitro. A S100 fraction, nrepared f r o m SV40 infected cells, was i n c u b a t e d at 32 ° for 35 rain. as described in the legend to t a b l e I (experiment 2), w i t h v a r i a b l e c o n c e n t r a t i o n s of Mg ++ a) in complete m e d i u m ( & - - - ~ ) ; b) o m i t t i n g GTP, CTP, and UTP ( & - - ~ ) ; c) o m i t t i n g the same a n d the energy generating system (@---@) ; or d) as in e) b u t w i t h o u t ATP (O--O). The S100 extract c o n t r i b u t e d 0.72 mg of protein per ml in the ass.ay.

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T h e i n f l u e n c e of t h e f o u r r i b o n u c l e o s i d e t r i p h o s p h a t e s w a s n e x t i n v e s t i g a t e d . T h a t of G T P , C T P a n d U T P w a s f o u n d to b e n e g l i g i b l e , b e c a u s e their addition was without major effect on either t h e g r o s s i n c o r p o r a t i o n of d N T P ' s b y t h e s y s t e m (fig. 1) o r t h e n a t u r e of t h e D N A p r o d u c t (see f o r example figure 7 in the accompanying paper). The i m p o r t a n c e of A T P a n d of a n A T P g e n e r a t i n g system, on the other hand, was capital : surprisingly, t h e a d d i t i o n of A T P a n d of a n e n e r g y g e n e r a t i n g s y s t e m d e c r e a s e d t h e i n c o r p o r a t i o n of d T M P 2 t o 3 f o l d ( t a b l e I a n d fig. 1). T h e e f f e c t of A T P o n t h e n a t u r e of t h e D N A l a b e l e d d u r i n g t h e i n c u b a t i o n in vitro, a n d o n t h e f a t e of t h e SV4,0 D N A c o m p o n e n t I u s e d as t e m plate in the assay, was therefore investigated. For that purpose, S¥40 DNA component I which had b e e n l a b e l e d in v i v o w i t h aep, w a s u s e d as t e m plate in an assay using [3H]dTTP to label the p r o d u c t , e i t h e r i n t h e p r e s e n c e o r a b s e n c e of 1 m M BIOCHIMIE, 1976, 58, n ° 9.

FIG. 2. - - Influence of A T P on the final size of the SV40 DNA template and product molecules. A $100 extract was i n c u b a t e d as described in t h e legend to t a b l e I, except t h a t GTP, CTP and UTP were omitted, and t h a t the SV40 DNA c o m p o n e n t I used as a template was labeled w i t h 32p. One assay was done in the presence of 1 mM ATP (left panels), the other in the absence of ATP (right panels). After 60 rain. incubation at 32 ° t h e reaction was stopped b y the a d d i t i o n of EDTA a n d p a r t f r o m each i n c u b a t i o n m i x t u r e was a n a l y z e d b y sucrose gradient c e n t r i f u g a t i o n at e i t h e r n e u t r a l pH (panels A and B) or a l k a l i n e pH (panels C and D). Symbols : O - - O : pmoles acid precipitable [3H]-dTMP ; 0 - - - 0 : cpm 32p DNA. The dashed line refers to the s e d i m e n t a t i o n of a sample of t h e 32p t e m p l a t e DNA w h i c h h a d not been i n c u b a t e d and was centrifuged in parallel.

in v i t r o w i t h [ 3 H ] - d T M P s e d i m e n t e d as f r a g m e n t e d m a t e r i a l at less t h a n 12S at n e u t r a l p H ( p a n e l B i n fig. 2) a n d at a p p r o x i m a t e l y 4S at a l k a l i n e p H ( p a n e l D).

I n t h e p r e s e n c e of A T P , o n t h e o p p o s i t e , m o s t of t h e 32p l a b e l e d D N A s e d i m e n t e d as u n i t l e n g t h

1106

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DNA m o l e c u l e s (left h a n d side panels in figure 2). ATP was t h e r e f o r e a p p a r e n t l y r e q u i r e d to maintain the i n t e g r i t y of the DNA t e m p l a t e molecules. ATP also c o n d i t i o n e d the f o r m of the DNA product obtained. A p p r o x i m a t e l y 50 p e r cent of the [3H]-dTMP labeled p r o d u c t m a d e in vitro by the S10.0, system in the p r e s e n c e of ATP s e d i m e n t e d at about 18S at neutral pH (open circles, p a n e l A in figure 2). Most of this label w a s in DNA comp o n e n t II molecules, as judged from its b e h a v i o r at aIkaline pH, but a definite amount w a s recov e r e d in DNA c o m p o n e n t I m o l e c u l e s (open circles, p a n e l C). H o w e v e r , these m o l e c u l e s l a c k e d s u p e r h e l i c i t y , s i n c e t h e y s e d i m e n t e d at s l o w e r t h a n 21S at neutral p H (panel A in figure 2) and since t h e i r density in the p r e s e n c e of e t h i d i u m b r o m i d e was h e a v i e r t h a n that of a Di~A c o m p o n e n t I m a r k e r (see fig. 3). Similarly, after i n c u b a t i o n w i t h the S100 e x t r a c t in t h e p r e s e n c e of ATP, the s e d i m e n t a t i o n p a t t e r n of the 32p labeled t e m p l a t e DNA was shifted at n e u t r a l p H f r o m a h o m o g e n e o u s 21S peak (dashed line in p a n e l A) to a h e t e r o g e n e o u s peak at approx i m a t e l y 18S (closed circles in p a n e l A). At alkaline pH, only 1/3 of the m o l e c u l e s still s e d i m e n t e d at 53S as a DNA c o m p o n e n t I m a r k e r (closed circles in panel C), w h i l e t h e r e m a i n d e r s e d i m e n t e d at 16-18S as DNA c o m p o n e n t II molecules. These results s h o w that in the p r e s e n c e of ATP, 1/3 of the t e m p l a t e DNA m o l e c u l e s w e r e c o n v e r t e d into r e l a x e d c i r c u l a r DNA m o l e c u l e s [2, 15, 36] and 2/3 into n i c k e d ( c o m p o n e n t II) DNA molecules. These results w e r e a s c e r t a i n e d by d e t e r m i n i n g the density d i s t r i b u t i o n in CsC1 in the p r e s e n c e of e t h i d i u m b r o m i d e of the DNA labeled in vitro using [SH] dTTP. T h e DNA p r o d u c t s labeled in the p r e s e n c e of ATP e x h i b i t e d a b i m o d a l distribution, w i t h 78 p e r cent of the p r o d u c t h a v i n g a density s i m i l a r to that of S¥40 D~N~A c o m p o n e n t II molecules, and 22 p e r cent a density h e a v i e r t h a n that of a u t h e n t i c c o m p o n e n t I m o l e c u l e s (fig. 3), c o r r e s p o n d i n g to r e l a x e d DNA c o m p o n e n t I molecules [36]. W h e n ATP w a s o m i t t e d f r o m the incubation m i x t u r e , the DNA s h o w e d a u n i m o d a l distribution, w i t h the totality of the label in the b a n d c o r r e s p o n d i n g to l i n e a r DNA m o l e c u l e s (not shown). T h e r e f o r e , although the a d d i t i o n of ATP s l o w e d t h e i n c o r p o r a t i o n of dNMP's (fig. 1), its p r e s e n c e w a s essential to p r e s e r v e the i n t e g r i t y of both t e m p l a t e and n e w l y s y n t h e s i z e d molecules. No intact c o m p o n e n t I m o l e c u l e s could be o b t a i n e d in the absence of ATP, w h e r e a s in the p r e s e n c e BIOCHIMIE, 1976, 58, n ° 9.

of ATP as m u c h as 2:5 p e r cent of the n u c l e o t i d e s i n c o r p o r a t e d , s e d i m e n t e d at 53S at pH 13 and b a n d e d w i t h a density c h a r a c t e r i s t i c s of covalently closed r e l a x e d c i r c u l a r DNA m o l e c u l e s in the p r e s e n c e of e t h i d i u m b r o m i d e .

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Fro. 3. - - Analysis of the in vitro labeled DNA by equilibrium centrifugation in CsCl in the presence of ethidium bromide. Part of a reaction mixture incubated for 60 min. at 32 ° with [3H'] dTTP (500 epm/pmole) was analyzed together with a 32p labeled SV40 DNA marker by equilibrium eentrifugation in the presence of ethidium bromide as described in M~aterial and Methods. After centrifugation, fractions were collected and assayed for TCA preeipitable radioactivity. S.ymbols : Q - - - O : 32p SV40 DNA marker ; (3--O : [3H] dTMP labeled material. The density of the marker DNA component I was 1.580 g/ee

D e g r a d a t i o n of the t e m p l a t e and n e w l y f o r m e d molecules, in the absence of ATP, w a s t e n t a t i v e l y a t t r i b u t e d to e n d o n u c l e a s e activity. T h e r e f o r e an attempt w a s m a d e at i n h i b i t i n g e n d o n u c l e a s e s by a d d i t i o n of KC1 or E. colt tRNA to the S100 system. F i g u r e 4 shows that in t h e p r e s e n c e of E. colt tRNA, o v e r a l l i n c o r p o r a t i o n of dTMP by a S10,0 e x t r a c t w a s decreased, but the final p r o d u c t was m u c h m o r e h o m o g e n e o u s and of l a r g e r size than that m a d e in the absence of tRNA. T h e effect of KC1 alone or c o m b i n e d w i t h tRNA on the overall i n c o r p o r a t i o n of dTMP are illustrated in figure 5. Both c o m p o u n d s l o w e r e d DNA p o l y m e r a s e activity, but to a different extent d e p e n d i n g u p o n the n a t u r e of the DNA used as t e m p l a t e in t h e assay. BuLk DNA p o l y m e r a s e activity, as m e a s u r e d w i t h a c t i v a t e d calf t h y m u s DNA, was not i n h i b i t e d by the a d d i t i o n of tRNA, and w a s p r o g r e s s i v e l y but slowly i n h i b i t e d by KC1 c o n c e n t r a t i o n s above 20 mM (triangles in figure 5). But dTMP i n c o r p o r a t i o n using SV40 DNA as t e m p l a t e was d e c r e a s e d 60 p e r cent by the m e r e a d d i t i o n of tI/NA and was extre-

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m e l y s e n s i t i v e t o a d d i t i o n of s a l t ( c i r c l e s i n f i g u r e 5). T h i s s h o w s t h a t t h e d e c r e a s e i n i n c o r p o r a t i o n i n t o SV40 D N A i n d u c e d in v i t r o b y t R N A a n d / o r KC1, is n o t so m u c h d u e to t h e i n h i b i t i o n of t h e D N A p o l y m e r a s e ( s ) i n t h e SI'00 e x t r a c t as to t h e i n h i b i t i o n of c o n t a m i n a t i n g e n d o n u c l e a s e s . T h e e f f e c t of v a r y i n g c o n c e n t r a t i o n s of KC1 o n t h e size of t h e in v i t r o l a b e l e d p r o d u c t a n d o n t h e f a t e of t h e SV40 c o m p o n e n t I t e m p l a t e w e r e also s t u d i e d . It w a s f o u n d t h a t t h e c o m b i n a t i o n of 3050 m M KCI t o g e t h e r w i t h 250 ~,g/ml of t R N A w a s o p t i m a l , as j u d g e d f r o m t h e a m o u n t of l a b e l e d d T M P i n c o r p o r a t e d p e r m g p r o t e i n of S100, a n d f r o m t h e p e r c e n t a g e of t h a t l a b e l w h i c h w a s r e c o v e r e d as u n i t l e n g t h D N A m o l e c u l e s at p H 13 ( n o t s h o w n ) . All s u b s e q u e n t e x p e r i m e n t s w e r e t h e r e c.P.,v,.

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m e n t i n g at 53S at p H 13 ( f r a c t i o n s 9 to 15 i n p a n e l s A-D), a n d t h a t i n u n i t l e n g t h D N A m o l e c u l e s s e d i m e n t i n g at a b o u t 18S at t h e s a m e p H ( f r a c t i o n s 22 to 28) v a r i e d l i n e a r l y w i t h t h e c o n c e n t r a t i o n of t e m p l a t e DNA, at l e a s t i n t h e r a n g e f r o m 20 t o 80 : ~ g / m l of DNA. T h e p r o p o r t i o n of l a b e l r e c o v e r e d as e o v a l e n t l y c l o s e d c i r c u l a r D N A m o l e c u l e s (53S) also i n c r e a s e d w i t h i n c r e a s i n g t e m p l a t e c o n c e n t r a t i o n . T h e a m o u n t of l a b e l i n f r a g m e n t e d D N A m a t e r i a l ( a b o u t 4S at p H 13) w a s h o w e v e r i n d e p e n d e n t of t h e c o n c e n t r a t i o n of t e m p l a t e DNA. Thus, the more concentrated the DNA template in t h e a s s a y , t h e g r e a t e r t h e p r o p o r t i o n of l a b e l i n closed circular DNA molecules.

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FIG. 4. -- Influence of tRNA on the size of the DNA made in vitro. The products of a 60 min. i n c u b a t i o n at 32 ° in the presence (panel B) or absence (panel A) of 250 p,g/ml of E. colt tRNA were analyzed together w i t h a ~2p labeled SV40 DNA m a r k e r b y c e n t r i f u g a t i o n at n e u t r a l pH. The assay was as described u n d e r Material a n d Methods using 2'.0 mM Tris pH 7.7, and no KC!. Symbols : O - - - O : 32p SV40 DNA m a r k e r ; O - - O : [:~H] dTMP labeled material.

f o r e p e r f o r m e d u s i n g t h e s e c o n c e n t r a t i o n s of KC1 a n d t R N A . I n a d d i t i o n , w e f o u n d , at t i m e s , a s l i g h t e n h a n c e m e n t of t h e l a b e l i n g of h i g h M.W. D N A w h e n u s i n g 1.5 m M p u t r e s c i n e i n t h e a s s a y . P u t r e s c i n e w a s t h e r e f o r e s y s t e m a t i c a l l y a d d e d i n all subsequent assays. Using these standardized conditions, the i n f l u e n c e of t h e c o n c e n t r a t i o n of t e m p l a t e D N A was next studied. An assay was carried out using 1.05 m g / m l of S100 p r o t e i n s a n d 10, 2.0, 40 a n d 80 ,~g/ml of SV40 D N A c o m p o n e n t I m o l e c u l e s . T h e D N A p r o d u c t l a b e l e d a f t e r 150 m i n . i n c u b a t i o n i n v i t r o w a s a n a l y z e d p a r t at p H 13 (fig. 6), a n d p a r t at n e u t r a l p H (fig. 7). As s e e n i n f i g u r e 6 (pf-'nel E), b o t h t h e a m o u n t of l a b e l r e c o v e r e d i n c o v a l e n t l y c l o s e d c i r c u l a r D~NA m o l e c u l e s s e d i BIOCHIMIE, 1976, 58, n ° 9.

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Fro. 5. - - Influence of KCt on the DNA polymerase actioity of a $100 extract. The effect of KC1 was studied in b o t h a s t a n d a r d 30 min. assay for DNA polymerase activity, using activated calf t h y m u s DNA as the t e m p l a t e (A), and in a 35 nfin. assay for SV40 DNA synthesis, using viral DNA c o m p o n e n t I as the t e m plate (O). Conditions for the l a t t e r were as in the legend to figure 4. Both assays used 48 ~tg of S100 extract proteins. Increasing c o n c e n t r a t i o n s of KCI were tested in the absence (left panel) or in the presence (right panel) of 250 g g / m l of E. cull tRNA. Control i n c o r p o r a t i o n in the absence of KC1 and tRNA were t a k e n as 100 per cent i n c o r p o r a t i o n : actual figures were 38 pmoles w i t h calf t h y m u s DNA and 10 pnmles w i t h SV40 DNA.

T h e D N A p r o d u c t w a s also a n a l y z e d at n e u t r a l p H (fig. 7). I n a g r e e m e n t w i t h t h e a b o v e r e s u l t s , t h e a m o u n t of l a b e l r e c o v e r e d i n D N A s e d i m e n t i n g f a s t e r t h a n 16S ( f r a c t i o n s 1'0-20 i n p a n e l s A-D of f i g u r e 7) v a r i e d l i n e a r l y w i t h t h e c o n c e n t r a t i o n of t e m p l a t e D N A ( p a n e l E). P a r t of t h e m a t e r i a l l a b e l e d in v i t r o n o w s e d i m e n t e d at 21S, i n t h e s a m e p o s i t i o n as a u t h e n t i c v i r a l D N A c o m p o n e n t I. T h i s is q u i t e d i f f e r e n t f r o m w h a t w a s o b s e r v e d i n t h e e x p e r i m e n t of f i g u r e 2 w h e r e t h e D N A w h i c h w a s 53S at p H 13 w a s a b o u t 18S at n e u t r a l pH. I n v i t r o l a b e l e d 21S D N A w a s r e p r o d u c i b l y o b t a i n e d w h e n u s i n g t h e a s s a y c o n d i t i o n s d e s c r i b e d a b o v e (see f i g u r e 8, a n d a c c o m p a n y i n g p a p e r ) . T h i s is p r o b a b l y t h e c o n s e q u e n c e of t h e i m p r o v e d a s s a y c o n d i t i o n s , i n p a r t i c u l a r of t h e u s e of t R N A , KC1 a n d putrescine.

1108

M. G i r a r d a n d coll.

f i g u r e 8. M n e u t r a l p H ( p a n e l A) t h r e e p e a k s of l a b e l e d m a t e r i a l w e r e i d e n t i f i e d at 21S, 18S a n d 4S r e s p e c t i v e l y . At a l k a l i n e p H ( p a n e l C) t h r e e p e a k s of m a t e r i a l w e r e i d e n t i f i e d at 53S, 16-18S a n d 4S. T h e s e s e d i m e n t a t i o n c o e f f i c i e n t s w e r e d e t e r m i n e d f r o m t h e s e d i m e n t a t i o n p r o f i l e of a 32p l a b e l e d v i r a l D N A m a r k e r c e n t r i f u g e d i n parallel.

T h e t i m e c o u r s e of S ¥ 4 0 D N A s y n t h e s i s i n v i t r o was then determined, using a template concentrat i o n of 50 ~ g / m l of SV40 D N A c o m p o n e n t I i n t h e p r e s e n c e of 46 m M KCI, 250 ~ g / m l of tR, NA a n d 1.5 m M p u t r e s c i n e (figure 8). At t h e e n d of e a c h incubation period, the reaction was stopped by t h e a d d i t i o n of E D T A a n d SDS. T h e D N A w a s extracted with phenol and precipitated with etha-

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Fro. 6. - - Influence of the concentration of SV$O DNA template on the nature of the in vitro product analyzed at alkaline pH. A s t a n d a r d incorporation assay was set as described in Material a n d Methods using 104 ~vg of a S100 extract protein, a n d v a r i a b l e a m o u n t s of t e m p l a t e SV40 DNA comp o n e n t I. B o t h dTTP and dGTP were labeled w i t h t r i t i u m . The reaction was stopped a f t e r 150' rain. i n c u b a t i o n at 32 ° b y t h e a d d i t i o n of EDTA a n d SDS, followed b y p h e n o l extraction. The ~Hlabeled DNA's were precipitated w i t h ethanol, resuspended into 0.1 × SSC, added w.it~h [32p] SV40 DNA1 as a m a r ker, a n d p a r t of each sample was analyzed b y sucrose g r a d i e n t centrifugation at a l k a l i n e pH : cpm 3H ((D--G) and cpm 32p ( - - - ) . The c o n c e n t r a t i o n s of SV40 DNA t e m p l a t e in the different assays were : panel A : 10 ,vg/ml ; p a n e l B : 2 0 . ~ g / m l ; p a n e l C : 40 ~ g / ml ; panel D : 80 l~zg/ml. The a m o u n t of 3H r a d i o a c t i v i t y recovered in 53S molecules (A--A) a n d i n 18S molecules ( A - - - A ) in t h e various gradients was plotted as a f u n c t i o n of t e m p l a t e conc e n t r a t i o n in p a n e l E.

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nol. P a r t of t h e p r e c i p i t a t e w a s a n a l y z e d b y s u c r o s e g r a d i e n t c e n t r i f u g a t i o n at n e u t r a l p H , w h e r e a s a n o t h e r p a r t w a s a n a l y z e d at a l k a l i n e p H . T h e s e d i m e n t a t i o n p a t t e r n of t h e p r o d u c t l a b e l e d a f t e r 300 r a i n . of i n c u b a t i o n in v i t r o is s h o w n as a n e x a m p l e i n t h e l e f t h a n d p a n e l s of BIOCHIMIE, 1976, 58, n ° 9.

a'o

DNA concentration(Hg/ml)

FIo. 7. - - Influence of the concentration of SV#O DNA template on the nature of the in vitro product analyzed at neutral pH. P a r t of the SV40 DNA synthesized in vitro in the preceding e x p e r i m e n t (fig. 6) was a n a l y z e d by sucrose g r a d i e n t c e n t r i f u g a t i o n at neut r a l p H . Symbols : O - - O : [3H] dTMP ; . . . . : [32p] DNA marker. The a m o u n t of 3H r a d i o a c t i v i t y recovered in material s e d i m e n t i n g f a s t e r t h a n 15S was plotted as a f u n c t i o n of t e m p l a t e conc e n t r a t i o n in p a n e l E (A--A),

T h e a m o u n t of r a d i o a c t i v i t y r e c o v e r e d i n t h e d i f f e r e n t p e a k s of t h e g r a d i e n t s a f t e r v a r i o u s t i m e s of i n c u b a t i o n w e r e p l o t t e d a g a i n s t t i m e t o d e t e r m i n e t h e t i m e c o u r s e of t h e in v i t r o l a b e l i n g of t h e t h r e e D N A s p e c i e s (fig. 8 p a n e l s B ( n e u t r a l p H ) a n d D ( a l k a l i n e p H ) ) . A f t e r s h o r t t i m e s of i n c u b a -

SV$O D N A s y n t h e s i s in vitro. lion, the labeling of small M.W. DNA f r a g m e n t s (4S) was p r e d o m i n a n t , but p l a t e a u e d r a p i d l y . A c c u m u l a t i o n of label into 18S m o l e c u l e s w a s m o r e sustained, but also e v e n t u a l l y p l a t e a u e d after 12,0 min. By contrast, after an i n i t i a l lag of 15-20 min., label a c c u m u l a t e d l i n e a r l y into closed c i r c u l a r D,NA m o l e c u l e s (21S, n e u t r a l pH, p a n e l B, and 53S, alkaline pH, p a n e l D). No e v i d e n c e for t r a n s i e n t a p p e a r a n c e of r e p l i c a t i v e i n t e r m e d i a t e (R.I.) molecules, w h i c h w o u l d h a v e been 2:5S at n e u t r a l pH [14, 18, 26] could be f o u n d even after the shortest times of i n c u b a t i o n e x a m i n e d . A

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1109

easily detectable at that stage using a c t i v a t e d calf t h y m u s DNA as template). T h e efficiency of the system after c o n c e n t r a t i o n by a m m o n i u m sulfate was c a l c u l a t e d f r o m the data o b t a i n e d in the t i m e course e x p e r i m e n t s h o w n in figure 8. A total of 24 pmoles each of dTMP and dGMP w e r e i n c o r p o r a t e d into SV40 DNA c o m p o n e n t I and c o m p o n e n t II m o l e c u l e s in t h e course of 300 min. Since t h e a m o u n t of t e m p l a t e SV4'0 DNA~ in the assay w a s 5 .~g, i.e. a p p r o x i m a t e l y 1.4 p m o l e DNA, it follows that on the average, about 75 d e o x y r i b o n u c l e o t i d e residues w e r e i n c o r p o r a t e d p e r m o l e c u l e of template DNA. T h e r a t i o of n e w l y s y n t h e t i z e d DNA to t e m p l a t e D NA w a s t h e r e f o r e only a p p r o x i m a tely of 0.6 p e r cent. On a m g of p r o t e i n basis, the system e x h i b i t e d a specific a c t i v i t y of 0.2 u n i t / m g w i t h SV4'0 DNA as t e m p l a t e (0.2 nmole d e o x y r i b o n u c l e o t i d e i n c o r p o r a t e d in 6'0 min. p e r mg of protein) w h i c h is about 15 times less t h a n that it e x h i b i t e d w i t h a c t i v a t e d calf t h y m u s DNA. The r e a s o n ~vhy the a c t i v i t y w i t h SV40 DNA was so l o w l~kely relates to the lack of free n a t u r a l 3'OH ends in the v i r a l DNA m o l e c u l e used as template, and to the slow rate at w h i c h free ends could be f o r m e d in the assay, for e x a m p l e as a c o n s e q u e n c e of e n d o n u c l e o l y t i c attack.

16S

DISCUSSION.

Fraction

number

Time

,60 a6o ~bo of i n c u b a t i o n (rain)

Fro. 8. - - Time course of SV~O DNA synthesis in vitro. A standard assay system (see Material and Methods) was incubated for varied length of times, in the presence of both [3H]-dTTP and [3H]-dGTP, after which the DNA ]ab,eled in vitro was a,na]yzcd as described in the legend to the preceding figures. The behavior of thc 300 min. sample is shown here as an illustration : part of that sample was analyzed at neutral pH (panel A), part at pH 13 (panerl C). The peaks labeled 21S in panel A, and 53S and 18S in panel B, refer to the sedimentation position of a 3:2p labeled SV40 DNA marker added to the sample prior to centrifugation. The number of counts recovered from each of these gradients as 21S, 18S and 4S material at neutral pH, and as 53S, 18S and 4S material at alkaline pH, are plotted as a function of time of incubation in panels B and D respectively. Note that different amounts of material were analyzed in the two sets of gradients.

It s h o u l d be p o i n t e d out that, t h o u g h m u c h h i g h e r t h a n in t h e n u c l e a r extract, the a c t i v i t y of the c y t o p l a s m i c f r a c t i o n ($100) of the i n f e c t e d ceils w a s quite l o w u n d e r the c o n d i t i o n s of the assay. F o r example, it w a s b a r e l y detectable at the step b e f o r e a m m o n i u m sulfate p r e c i p i t a t i o n (although gross DNA p o l y m e r a s e a c t i v i t y w a s BIOCHIMIE, 1976, 58, n ° 9.

Since t h e r e p l i c a t i o n of SV40 DNA t~l~es p l a c e in the nucleus of the i n f e c t e d cell in vivo, and since elongation and t e r m i n a t i o n of SV40 DNA r e p l i c a t i o n can be c a r r i e d out by isolated n u c l e i in vitro [5, 6, 24], the obvious a p p r o a c h to the o b t e n t i o n of a soluble, cell free r e p l i c a t i o n system for SV40, should h a v e been to start f r o m the n u c l e a r f r a c t i o n of SV40 i n f e c t e d cells. H o w e v e r , the e x p e r i m e n t s r e p o r t e d h e r e show that the n u c l e a r e x t r a c t f r o m S¥40 i n f e c t e d cells h a d v e r y little detectable DNA p o l y m e r a s e a c t i v i t y w h e n s u p p l e m e n t e d in vitro w i t h SV40 DNA comp o n e n t I m o l e c u l e s as template. By contrast, the c y t o p l a s m i c e x t r a c t f r o m the same cells d i s p l a y e d a definite ability to i n c o r n o r a t e d e o x y r i b o n u c l e o tides u n d e r the same conditions. T h e lack of a c t i v i t y of the n u c l e a r f r a c t i o n was not due to t h e loss or i n a c t i v a t i o n of n u c l e a r DNA p o l y m e r a s e s , since, w h e n assayed w i t h an activated DNA template, t h e n u c l e a r e x t r a c t s w e r e at least 50 p e r cent as active as the c y t o p l a s m i c extracts (per mg of p r o t e i n ) . F o r example, w i t h a c t i v a t e d calf t h y m u s DNA as a template, the n u c l e a r (N100) f r a c t i o n f r o m the e x p e r i m e n t s h o w n in table I displayed an a c t i v i t y of 1.82 units DNA p o l y m e r a s e p e r mg of protein, w h e r e a s the

1110

M. Girard a n d coll.

c y t o p l a s m i c (S100) f r a c t i o n d i s p l a y e d an activity of 2.5,0 u n i t s / r a g . Still, w i t h S V4,0 DNA c o m p o n e n t I as template, t h e same n u c l e a r f r a c t i o n w a s 6-10 t i m e s less a c t i v e t h a n t h e c y t o p l a s m i c f r a c t i o n . A possible e x p l a n a t i o n for this lack of activity is that c r i t i c a l e n z y m e s or factors r e q u i r e d for the synthesis of SV40 DNA in vitro leaked out f r o m the n u c l e i d u r i n g the p r e p a r a t i o n of t h e cytoplasm i c extracts. Several n u c l e a r e n z y m e s are k n o w n to leak out f r o m nuclei w h e n aqueous p r o c e d u r e s are used for t h e f r a c t i o n a t i o n of the cell. F o r example, class C m a m m a l i a n RNA p o l y m e r a s e is p r e f e r e n t i a l l y r e c o v e r e d in the c y t o p l a s m i c fraction of cell extracts [12]. Also, elongation of nascent DNA chains by isolated n u c l e i f r o m HeLa cells r e q u i r e s t h e p r e s e n c e of the soluble p r o t e i n f r a c t i o n f r o m t h e c y t o p l a s m [10, 16, 17]. Similarly, most of the synthesis of v i r a l D~NA c o m p o n e n t I w h i c h can take p l a c e in vitro in a SV40 or polyom a v i r u s i n f e c t e d cell lysate, is lost w h e n the n u c l e i are s e p a r a t e d f r o m the c y t o p l a s m i c c o m p o nents, but r e s t o r e d w h e n the nuclei are i n c u b a t e d in the p r e s e n c e of the soluble p r o t e i n f r a c t i o n f r o m t h e c y t o p l a s m [5, 8, 21, 24~. It is l~nown also that SV4,0 e a r l y proteins, such as T antigen, w h i c h are l o c a t e d i n s i d e t h e cell nucleus in vivo, are easily r e c o v e r e d f r o m the c y t o p l a s m i c f r a c t i o n of cell e x t r a c t s in vitro [23, 30]. T h e reason w h y the n u c l e a r extracts f r o m S¥40 i n f e c t e d cells w e used failed to r e p l i c a t e e x o g e n o u s l y a d d e d SV40 DNA, could t h e r e f o r e be that t h e y h a d lost, into the cytoplasm, one or several of the factors r e q u i r e d to c a r r y out the in vitro synthesis of c o v a l e n t l y closed c i r c u l a r DNA molecules. A n o t h e r possibility is that the e n v i r o n m e n t in w h i c h the n u c l e a r e x t r a c t s w e r e tested l a c k e d some c o f a c t o r or activ a t o r w h i c h is r e q u i r e d for the i n i t i a t i o n of v i r a l DNA r e p l i c a t i o n , and the n a t u r e of w h i c h is presently u n k n o w n . Since only the c y t o p l a s m i c f r a c t i o n of the cell s h o w e d a c t i v i t y w i t h SV4,0 DNA as a template, w e c o n c e n t r a t e d on the study of that activity. I n c o r p o r a t i o n w i t h that system w a s r a t h e r r e m a r k a b l e , b o t h by the n a t u r e of the l a b e l e d p r o d u c t and by t h e p r o l o n g e d d u r a t i o n of t h e i n c o r p o r a t i o n . Indeed, the $100 e x t r a c t w a s able to i n c o r p o r a t e the m o n o p h o s p h a t e m o i e t y of the four d e o x y r i b o n u c l e o s i d e t r i p h o s p h a t e s into a c o v a l e n t l y closed, c i r c u l a r DNA p r o d u c t w h i c h , e x c e p t for the s u p e r h e l i c i t y of t h e molecule, w a s i n d i s t i n g u i shable f r o m a u t h e n t i c SV4'O DNA r This r e a c t i o n could be m a d e to last at a c o n s t a n t rate for at least 300. rain., close to 6~0 p e r cent of t h e DNA p r o d u c t b e i n g r e c o v e r e d as r e l a x e d DNA eom~)onent I molecules, and about 30 p e r cent as D N 4 component II (see fig. 8).

BIOCHIMIE, 1976, 58, n ° 9.

The only obvious r e q u i r e m e n t for the r e a c t i o n to last that long was that for a constant supply of ATP. In the absence of ATP, all t e m p l a t e m o l e c u l e s w e r e d e g r a d e d to small M.W. fragments, demonst r a t i n g e x t e n s i v e attacks by endonucleases, and the r e a c t i o n s t o p p e d progressively. In the p r e s e n c e of ATP, on the o t h e r hand, the t e m p l a t e m o l e c u l e s did not suffer m o r e t h a n a few single s t r z n d breaks, since not m o r e t h a n 60 p e r cent (fig. 2) and usually only 25-30 p e r cent (see accom~)anying paper) w e r e r e c o v e r e d as DfNA c o m p o n e n t II molecules. None a p p e a r e d to be f r a g m e n t e d to small M.W. fragments. This suggests that in the p r e s e n c e of ATP, e i t h e r the a c t i v i t y of the e n d o n u c l e a s e s of the e x t r a c t was i n h i b i t e d , or, rather, r a n d o m single strand attacks of the c i r c u l a r template DNA m o l e c u l e s by the e n d o n u c l e a s e s of the e x t r a c t still o c c u r r e d , but w e r e q u i c k l y r e p a i r e d by the cell DNA ligase. It is k n o w n that m a m m a l i a n cell DNA ligase is d e p e n d e n t on ATP [25], w h i c h w o u l d e x p l a i n w h y h i g h levels of ATP w e r e c o n s t a n t l y r e q u i r e d to p r e v e n t excess d e g r a d a t i o n of the DNA. This, in turn, suggests that the a c t i v i t y of the $1.00 system is mostly that of a r e p a i r - l i k e p r o c e s s i n v o l v i n g the breakdo'wn and resynthesis of t e m p l a t e DNA molecules~ but not net synthesis of DNA. That this is i n d e e d the case will be d e m o n s t r a t e d in the f o l l o w i n g paper.

Acknowledgments. This work was supported in part by the Ddldgation GGn~rale h la Recherche Scientifique et Technique and the Commissariat h l'Energie Atomique. R~su~. Dans le but d'~tudier la r~plication du DNA dans un systGme acellulaire d~riv~ de ce]lu]es animales en cultures, nous avons choisi 4'~tudier la synthGse du DNA du SV~0 en extraits de cellul.es CV~ infeetdes par ]e virus. On a compar6 l'.acti~it~ d'e~traits eytoplasmiques et d'extraits nucl~aires. En presence de DNA de thymals de veau activ6 utilis~ comme matrice, les deux extraits manifestent des activit~s DNA polymGrase comparables. Par contre en prGsence de DNA de SV40 forme I comme matrice, seul l'extrait cytoplasmique manifeste une aetivit~ significative. Nons avons donc eoneentr6 notre attention sur le syst~me cytoplasmique. Ce syst~me catalyse l'incorporation des 4 d~soxyrihonuclGotides dans un produit qui possGde ]es mdmes propriGt~s de s~dimentation, taut h pH neutre qu'h pH alcalin, que les forints I e t II du DNA viral. Les formes I synth~tis~es in vitro sont toutefois d~pourvues de superh~licit~. Le systGme est exigeant en Mg++, en DNA, en dGsoxyribonuclGotides, en &TP et en syst~me g~n~rateur d'Gnergie. La cin~tique d'incorporation de dTMP darts ]e DNA forme I montre un temps de latence d'environ 15 mn, aprGs quoi elle devient lin~aire. Elle se maintient h taux constant jusqu'h plus de 5 heures h 32°C. La cinGtique d'incorporation de dTMP dans le DNA forme I I n e montre pas de latence. Elle atteint un plateau aprGs quelque 2 heures h 32°C. Ceci suggGre qu¢

SV40 DNA

synthesis

les molecules de f o r m e II sont des pr6curseurs des molecules de f o r m e I. L'ATP cst indispensable pour m a i n t e n i r l'int~grit~ des DNAs d u r a n t la r6action. REFERENCES. 1. Bolden, A., Aucker, J. & Weissbach, A. (1975) J. Virol., 16, 1584:-15.9,2. 2. Champoux, J. J. & Dulbeeco, R. (1972) Proc. Nat. Acad. Sci. U.S., 69, 143-146. 3. Chang, L. M. S. & Bollnm, F. J. (1971) J. Biol. Chem., 246, 5835-5'837. 4. Chiu, R. W. & Baril, E. F. (1975) J. Biol. Chem., 250, 7951-7957. 5. Depampbilis, M. L. & Berg, P. (1975) J. Biol. Chem., 250, 4348-4354. 6. Depamphilis, M. L., Beard, P. a Berg, P. (1975) J. Biol. Chem., 250, 43i40~4347. 7. Francke, B. a Eckhart, W. (1973) Virology, 55, 127135. 8. Francke, B. a Hunter, T. (1975) J. Virol., 15, 9~7-107. 9. Fried, M. (1970) Virology, 40~ 605-617. 10. Hershey, H. V., S¢i~eber, J. F. ~, Mueller, G. C. (1973) Eur. J. Biochem., 34, 383-3:94. 11. Hirt, B. (1967~ J. Mol. Biol., 26, 365~-369. 12. Hossenlop, P., Wells, D. a Chambon, P. (1975) Eur. J. Biochem., 58, 2~3,7-251. 13. Hunter, T. a Francke, B. (1974) J. Virol., 13, 125139. 14. Jaenisch, R., Mayer, A. ~ Levine, A. J. (1971) Nature New Biol., 233, 72-75. 15. Keller, W. (1975) Proc. Nat. Acad. Sci. US., 72, 25502554. 16. Kidwell, W. R. (1972~) Biochem. Biophys. Acta, 269, 51-61. 17. Kidwell, W. R. a Mueller, G. C. (1969) Biochem. Biophys. Res. Commun., 36, 756-763.

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18. Levine, A. J , Kang, H. S. a Billheimer, F. E. (1970) J. Mol. Biol., 50, 549-568. 19. M.agnusson, G. (1973) J. Virol., 12, 609-615. 20. Magnusson, G., Pigiet, V., ~Vinnaeker, E. L., Abrams, R. a Reichard, P. (1973) Proc. Nat. Acad. Sci. U.S., 70, 412-415. 21. Otto, B. a Reichard, P. (1975) J. Virol., 15, 259-267. 22. Pages, J., Manteuil, S., Stehelin, D., Fiszman, M., Marx, M. ~, Girard, M. (1973) J. Virol., 12, 99-107. 23. Prives, C., Aviv H., Gilboa, E., Winocour, E. ,~ Revel, M. (1975) In vitro transcription and translation of viral genomes. A. L. Haenni & Beaud, G. ed. INSERM, Paris, 47, 305-312. 24. Quasba, P. K. (1974) Proc. Nat. Acad. Sci. U.S., 71, 104-5-1049. 25. Sambrook, J. & Shatkin, A. J. (1969) J. Virol., 4, 719-726. 26. Sebring, E. D., Kelly, T. J., Thoren, M. M. & Salzman, N. P. (1971) J. Virol., 8, 478-490. 27. Sedwick, W. D., Shu-Fong Nuang, T. & Korn, D. (1972) J. Biol. Chem., 247, 5026-5033. 28. Spadari, S. & x~Veissbach, A. (1974) J. Mol. Biol., 86, 11-20. 29. Tegtmeyer, P. (1972) J. Virol., 10, 591-598. 30. Tegtmeyer, P., Schwartz, M., Collins, J. K. & Rundell, K. (1975) J. Virol., 16, 168-178. 31. Weissbach, A. (1975) Cell., 5, 101-108. 32. Weissbach, A., Schlabach, A., Fridlender, B. Bolden, A. (1971) Nature Ne~u, Biol., 23'1, 167-170. 33. Wickner, R. B., Ginsberg, B., Berkower, I. & Hurwitz, J. (1972) J. Biol. Chem., 247, 489-497. 34. Winnacker, E. L., Magnusson, G. & Reichard, P. (1972) J. Mol. Biol., 72, 523-537. 35, Wintersberger, U. a Wintersberger, E. (1975) J. Virol., 16, 1095-1100. 36. Yu, K, & Cheevers, W. P. (1976) J. Virol., 17, 402-414.

In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells.

BIOCHIMIE, 1976, 58, 1101-1111. In vitro synthesis of simian virus 40 DNA. I. - Synthesi~ b y a soluble extract from infected CV1 cells. M a r c GIRA...
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