BIOCHIMIE, 1976, 58, 1113-1122.

In vitro synthesis of simian virus 40 DNA. II. - Evidence

for a repair m e c h a n i s m .

Louise MARTY, Chantal CAJEAN, F r a n c o i s e SUAREZ a n d M a r c GIRARD ~ . Unitd de Phgsiologie 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 , 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 . - - The t e c h n i q u e of density labeling of DNA b y BrdU was used to characterize the m a t e r i a l synthesized in vitro by cytoplasmic extracts of SV40 infected cells i n c u b a t e d in the presence of s i m i a n v i r u s 40 (SV40) DNA c o m p o n e n t I molecules (Girard et at, Biochimie, this volume). In a first experiment, the t e m p l a t e was labeled b e f o r e h a n d in vivo using [14C]-BrdU, and the in vilro i n c u b a t i o n was carried out in the presence of [3H]-dGTP a n d [3H]-dTTP. In a second experiment, the t e m p l a t e was labeled in vivo w i t h 32p, a n d the in vitro i n c u b a t i o n was in the presence of [3H]-dGTP and BrdUTP. After digestion w i t h the r e s t r i c t i o n endonuclease Hind II d- III, the f r a g m e n t s f r o m the end products of the r e a c t i o n 'were a n a l y z e d by density g r a d i e n t centrifugation, at pH 7 a n d pH 13. In b o t h e x p e r i m e n t s the DNA product molecules h a d the same density as the respective DNA templates. Cellular enzymes seem to be responsible for lhis in vitro synthesis of DNA, since cytoplasmic extracts f r o m uninfected cells were a h n o s t as active as those f r o m SV40 infected cells. The system was proved efficient in the conversion of molecules (component II DNA molecules) to covalently closed circular D N A - m o l e c u l e s (relaxed c o m p o n e n t I molecules). The use of DNA complexed w i t h h i s t o n e s did not i m p a r t v i r a l specificity to the system. It is concluded t h a t the cytoplasmic extract is only capable of supporting the r e p a i r synthesis of added v i r a l DNA.

INTRODUCTION. I n t h e a c c o m p a n y i n g p a p e r [9], w e d e m o n s t r a t e d t h a t t h e 100,0,00 g s u p e r n a t a n t ($1'00) of a c y t o p l a s m i c e x t r a c t f r o m S ¥ 4 0 i n f e c t e d cells c a t a l y z e d t h e D N A - d e p e n d e n t 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 cleotides in vitro. When such an extract was supp l e m e n t e d w i t h SV40 D N A f o r m I m o l e c u l e s as template, the four deoxyribonucleoside-triphosphates were incorporated into a product which w a s i n d i s t i n g u i s h a b l e f r o m a u t h e n t i c SV40 D N A c o m p o n e n t II m o l e c u l e s a n d r e l a x e d c o m p o n e n t I molecules. The reaction was linear with time for as l o n g as 5 h r s at 32°C. T h e s y s t e m w a s h i g h l y dependent on added ATP and an ATP generating system. T h e q u e s t i o n w e p o s e d is w h e t h e r t h i s s y s t e m s u p p o r t s t h e a c t u a l r e p l i c a t i o n of S ¥ 4 0 DNA, o r o n l y p r o m o t e s t h e i n v i t r o l a b e l i n g of D N A m o l e cules through a repair-like mechanism involving b r e a k d o w n a n d r e s y n t h e s i s of t h e t e m p l a t e . I n o t h e r w o r d s , t h e q u e s t i o n is w h e t h e r t h e s y s t e m c a n c a t a l y z e n e t s y n t h e s i s of SV40 D N A o r not. S e m i - c o n s e r v a t i v e r e p l i c a t i o n of D N A i n a cellfree system can easily be differentiated from To w h o m all correspondence should be addressed.

r e p a i r - l i k e p r o c e s s e s b y t h e use of d e n s i t y l a b e l i n g i n v i t r o [6, 19]. If a c t u a l D N A r e p l i c a t i o n w e r e to be involved in the system, the density marker s h o u l d a l l o w c l e a r d i s t i n c t i o n of t h e n e w l y s y n thesized daughter strands from the parental s t r a n d s o n t h e b a s i s of t h e i r r e s p e c t i v e d e n s i t y . O n t h e o t h e r h a n d , if o n l y D N A r e p a i r s y n t h e s i s were taking place, no density difference should be expected between parental and daughter DNA s t r a n d s , s i n c e p a r t i a l s u b s t i t u t i o n of n u c l e o t i d e s in a template DNA molecule would not grossly a f f e c t its b u o y a n t d e n s i t y . T h e e x p e r i m e n t w a s first p e r f o r m e d b y u s i n g SV40 D N A m o l e c u l e s l a b e l e d i n v i v o w i t h [14C~B r d U , u s i n g [ ~ H ! - d T T P as a l a b e l i n v i t r o . T h e d e n s i t y of t h e f i n a l i n v i t r o D N A p r o d u c t w a s c o m p a r e d t o t h a t of t h e t e m p l a t e , b y e q u i l i b r i u m c e n t r i f u g a t i o n a n a l y s i s , a f t e r f r a g m e n t a t i o n of t h e DNA molecules. The converse experiment, using u n l a b e l e d SV4,0 D N A m o l e c u l e s as a t e m p l a t e a n d B r d U T P i n l i e u of d T T P to l a b e l t h e p r o d u c t w a s also p e r f o r m e d . T h e s p e c i f i c i t y of t h e s y s t e m w a s f i n a l l y t e s t e d using cytoplasmic extracts from uninfected cells o r f r o m c e l l s i n f e c t e d w i t h t h e tsA3'0 m u t a n t of SV40 [29]. T h e c o m b i n e d r e s u l t s of t h e s e e x p e r t 76

L. Martg a n d coll.

1114

m e n t s suggest that the m a j o r i t y of the SV40 DNA synthesis catalyzed in vitro by the c y t o p l a s m i c system results from an extensive b r e a k d o w n a n d resynthesis of D NA molecules w h i c h is not virusspecific. MATER~AL A~ND METHODS. The growth of CV 1 cells, t h e i r i n f e c t i o n by SV40, the p r e p a r a t i o n of DNA free S100 extracts a n d t h e i r assay in vitro were described i n the accomp a n y i n g p a p e r [91. The i n f e c t i n g virus was either w i l d type (wt) or tsA30 m u t a n t of SV40 [29] o b t a i n e d from P. Tegtmeyer. Both s t r a i n s were g r o w n at low m u l t i p l i c i t y of i n f e c t i o n (1'0-~ p f u / cell) from plaque-purified stocks.

Preparation of template molecules. C o m p o n e n t I molecules of SV4,0 DNA, unlabeled or labeled w i t h 32p, were p r e p a r e d as desc r i b e d [9]. Density labeled c o m p o n e n t I molecules were o b t a i n e d from SV40~ infected cells labeled for 48 hrs before lysis w i t h 5 ,~g/ml of 2-[14C]-5-bro m o d e o x y - u r i d i n e (BrdU, New E n g l a n d Nuclear) added at a final specific activity of 15-20 .uCi/mg. The BrdU labeled DNA c o m p o n e n t I molecules, extracted b y the Hirt p r o c e d u r e [16], were first purified by two successive c e n t r i f u g a t i o n s i n CsCl i n the p r e s e n c e of e t h i d i u m bromide. The dye was r e m o v e d and the BrdU substituted DNA was isolated by a f u r t h e r CsC1 gradient c e n t r i f u g a t i o n i n the absence of e t h i d i u m bromide. The m a t e r i a l was dissolved i n 4 inl CsC1 adjusted w i t h T E buffer (1~0-3 M Tris pH 7.4,1'0 -4 M E D T A ) t o a d e n s i t y of 1.720 and centrifuged for 90 hours at 35;0'00 rpm, 20°C, i n the SW 50.1 Spineo rotor. The DNA w i t h a density of greater t h a n 1.710 g / c c was pooled, dialyzed against TE buffer, p r e c i p i t a t e d w i t h ethanol a n d stored at a c o n c e n t r a t i o n of 500 ~g of D;NA per m l in TE buffer at - - 8 0 ° C . It was diluted to 200 L~tg/ml w i t h Tris Mg ÷+ p r i o r to use, as d e s c r i b e d [9]. Component II nmlecnles were p r e p a r e d from ~2p labeled purified DNA compon e n t I molecules treated w i t h 5 × 10 -4 .~g/ml RNase-free DNase (Worthington) as described previously [203. Resulting form II DNA was separated from r e m a i n i n g form I DNA by e q u i l i b r i u m centrifugation in CsC1 i n the p r e s e n c e of e t h i d i u m bromide. The D'Nase t r e a t m e n t was calculated from a p r e l i m i n a r y k i n e t i c experiment, to yield 85 p e r cent DNA form II. The Poisson d i s t r i b u t i o n for this percentage c o n v e r s i o n implies that most resulting form II molecules had u n d e r g o n e 1 to 3 single s t r a n d breaks.

BIOCHIMIE, 1976, 58, n ° 9.

SV40 m i n i c h r o m o s o m e s were r e c o n s t i t u t e d from 3up labeled SV4.0 DNA c o m p o n e n t I and calf thymus histones devoid of h i s t o n e F1, as described by Oudet et al [24]. The calf t h y m u s histones (gift from P. S a u t i ~ r e s ) w e r e e x t e m p o r a n e o u s l y dissolved into a m i n i m a l volume of 10 -3 N HC1 a n d added w i t h 32p-labeled SV40 DNA c o m p o n e n t I molecules i n 100 mM Tris HC1 pH 7.5, 2 mM DTE, 2 mM EDTA, 10 mM bisu]fite. Salt c o n c e n t r a t i o n was adjusted to 2 M w i t h respect to NaC1, and the m i x t u r e (100 !~g/ml 32P-DNA, 188 ~ g / m l histones) was dialyzed against decreasing concent r a t i o n s of NaC1 : 2 M for 2 hrs, 1.5 M for 16 hrs, 1 M for 24 hrs, 0.75 M for 12 hrs, 0.5 M for 23 hrs, 0.25 M for 16 hrs. It was finally c o n c e n t r a t e d by v a c u u m dialysis. All dialysis solutions c o n t a i n e d d i t h i o e r y t h r i t o l (2 mM), EDTA (2 mM), a n d Na bisulfite (10 raM). The resulting m i n i c h r o m o s o m e s s e d i m e n t e d at m o r e t h a n 60 S at n e u t r a l pH. It was ascertained that, after d e p r o t e i n i z a t i o n , the DNA they c o n t a i n e d was still 53S at alkaline pH a n d 21S at n e u t r a l pH (results not shown).

Analysis of the i n vitro labeled DNA. The DNA was analyzed by c e n t r i f u g a t i o n t h r o u g h sucrose gradients i n 1 M NaC1 at n e u t r a l pH or at pH 13, as described i n the a c c o m p a n y i n g p a p e r [9]. T r e a t m e n t w i t h r e s t r i c t i o n e n d o n u c l e a s e s from

Hemophilus Influenzae (Hind II -t- III) was i n 6.6 mM Tris HC1 pH 7.4, 6.6 mM MgC12, 6 mM .~ mercaptoethanol, 50 mM NaC1 (4, 22-23). The enzymes were a gift from R. Monier and P. Nardeux. The r e a c t i o n was stopped, after 2 hrs i n c u b a t i o n at 37°C, by the a d d i t i o n of 5 mM EDTA. The digested m a t e r i a l was m a d e 1 p e r cent w i t h respect to SDS, i n c u b a t e d for 30 min. at 37°C, then analyzed by electrophoresis t h r o u g h 17 × 1.1 cm, 4 per cent p o l y a c r y l a m i d e gels for 15 hrs at 7 mA per gel [21]. The gels were frozen a n d cut into 2 m m t h i c k slices. Each slice was i n c u b a t e d for 15 hrs at 20°C in 1 ml soluene (New E n g l a n d Nuclear) before c o u n t i n g i n 10 ml s c i n t i l l a t i o n fluid (Omnifluor, New E n g l a n d Nuclear). The Hind II ~- III digests equilibrium centrifugation in up to a final d e n s i t y of 1.72 Fris..HC1 pH 7.4,10 -4 M EDTA. for 70 hrs at 37,000 rpm, 2,0°C, rotor.

were analyzed b y n e u t r a l CsC1 m a d e in 5 ml of 10 -2 M Centrifugation was i n the Spinco 50 Ti

Analysis by e q u i l i b r i u m e e n t r i f u g a t i o n in alkaline CS2SO 4 was a c c o r d i n g to either of the following methods. I n m e t h o d A [19!, the Hind II -J- III digest was extracted w i t h phenol, p r e c i p i tated w i t h ethanol, and r e s u s p e n d e d into 10 mM

Repair s y n t h e s i s of SV40 D N A in vitro. Tris-HC1 pH 7.8, 5 mM EDTA, 250 mM NaC1, after w h i c h the sample w a s diluted to a final v o l u m e of 4 ml w i t h 8.0 mM NaOH, 20 mM Na3PO4, before being a d d e d w i t h CseSO 4 to a final density of 1.41 g / c c . C e n t r i f u g a t i o n w a s for 90 hrs at 3,0,00,0 rpm, 20°C, in the SW 5'0.1 r o t o r of the Spinco. In m e t h o d B, the H i n d II -t- III digest was diluted to 7 ml w i t h 10 mM Tris pH 8.4), 1 mM

A

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mic extract from SV40 infected cells was incubated for 160 rain. at 32 ° in a volume of 1 ml with 40 ~g/ml SV40 DNA component I molecules which had been labeled in oivo either with 32p or with [14C]-BrdU. Final concentrations were 20. inM Tris-HC1 pH 7.7, 5 mM DTE, 6.5 mM MgC1.,, 100 !xM each of dATP and dCTP, 50 ttM each of I3H]-dTTP and [3H]-dGTP (both at 1500 cpm/pmole) 6 inM PEP, 1 mM ATP, 5 i~g/ml pyruvate kinase, 1.5 mM putrescine, 250 ~g/ml E. colt tRNA and 30 mM KC1. In addition, the extract brought 6.6 mM Tris HC1, 2 mM 2-~-mercaptoetbanol, 0.66 mM EDTA, 2.5 per cent glycerol, and 1 mg/ml of cellular proteins. The reaction was stopped by the addition of 4 vol. of 0.2 M EDTA, followed by 1 vol. of 10-2 M Tris-HC1 pH 7.4, 2 mM EDTA, 0.6 per cent SDS. The labeled material was extracted three times with phenol-chloroform (1:1), precipitated with ethanol, and analyzed by sucrose gradient centrifngation at neutral pH (left panels). Part of the material from the fractions of the gradients shown by the brackets in panels A and C was pooled, ethanol precipitated and further analyzed by sucrose gradient centrifugation at alkaline pH (right panels). Symbols : Q - - - O : 3H ; O--O : 32p ; /~--A : 14C.

EDTA, 200 mM NaC1. A 1/10, v o l u m e of 1 N NaOH w a s t h e n added, after w h i c h the solution was adjusted to a density of 1..42 g / c c w i t h Cs2SO ~. C e n t r i f u g a t i o n was for 80 hrs at 27;0'00 rpm, 15°C, in the Spinco 50 Ti rotor.

BIOCHIMIE, 1976, 58, n ° 9.

1115 RESULTS.

D e n s i t y labeling o[ the DNA in vitro. In a first series of e x p e r i m e n t s , a c y t o p l a s m i c f r a c t i o n ($100) f r o m SV4,0 i n f e c t e d cells w a s incubated in a s t a n d a r d assay using [~4C]-BrdU labeled SV40 DNA c o m p o n e n t I m o l e c u l e s as template. Synthesis of DNA w a s m o n i t o r e d by the i n c o r p o r a t i o n of [aH]-dTTP and [aHI-dGMP. At t h e end of the i n c u b a t i o n p e r i o d , the DNA in t h e incubation m e d i u m was analyzed 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 n e u t r a l p H (fig. 1, p a n e l C). T h e m a j o r i t y of the in vitro aH labeled DfNA sedim e n t e d at 16S or more, in a p a t t e r n i n d i s t i n guishable from that of t h e original 14C labeled t e m p l a t e molecules. At least h a l f of both the product and the t e m p l a t e w e r e u n d e r the f o r m of 21S DNA molecules. As a c o m p a r i s o n , 32p labeled (light) SV40 DNA m o l e c u l e s w e r e used as a t e m p l a t e in a p a r a l l e l assay, u s i n g the same S10,0 e x t r a c t and i d e n t i c a l c o n d i t i o n s of i n c u b a t i o n (fig. 1, p a n e l A). Comparison of panels A and C s h o w s that i d e n t i c a l sedim e n t a t i o n profiles of t h e p r o d u c t s , and s i m i l a r extents of in vitro i n c o r p o r a t i o n w e r e f o u n d using e i t h e r template. In both e x p e r i m e n t s , part of the DNA m o l e c u l e s s e d i m e n t i n g at m o r e than 16S at n e u t r a l pH (brackets in panels A and C) w a s analyzed at pH 13 by sucrose g r a d i e n t c e n t r i f u g a t i o n (fig. 1, right panels). T h e m a j o r i t y of both templates, w h e t h e r light DNA labeled w i t h a2p (open circles, p a n e l B) or h e a v y DNA labeled w i t h [14C]-BrdU (open triangles, panel D) w e r e r e c o v e r e d u n d e r the f o r m of 53S DNA ( c o m p o n e n t I molecules). T h e major i t y of the n e w l y labeled DNA, in e i t h e r assay (closed circles) w a s also r e c o v e r e d as 53S DNA molecules. T h e absence of small M.W. f r a g m e n t s d e m o n s t r a t e s that the few DNA c o m p o n e n t II m o l e c u l e s f o r m e d in the assay w e r e m a d e of unit t e n t h strands. T h e label in DNA c o m p o n e n t I and c o m p o n e n t II molecules w a s f u r t h e r analyzed, using t h e restriction enzymes H i n d II -b III f r o m H e m o p h i l u s influenzae, as d e s c r i b e d by Nathans and D a n n a [22, 23]. T h e r e s u l t i n g f r a g m e n t s w e r e analyzed by electrophoresis on polyacrylamide gels. F i g u r e 2 shows that the r a t i o of aH to 14C (first e x p e r i m e n t ) or aH to a2p (second e x p e r i m e n t ) was the same in all the fragments. This i m p l i e s a uniform labeling along the length of the in vitro labeled material, w h e t h e r the t e m p l a t e used w a s ,;ubstituted w i t h B r d U (upper panel) or s i m p l y labeled w i t h a2p (lower panel). S i m i l a r observa-

L. Martg and coll.

1116

t i o n s h a v e also r e p e a t e d l y b e e n m a d e u s i n g u n l a b e l e d SV4,0 D N A m o l e c u l e s as t e m p l a t e . So far, a n d e v e n a f t e r t h e s h o r t e s t t i m e s of i n c u b a t i o n tested, w e h a v e not b e e n a b l e to d e t e c t t h e p r e f e r e n t i a l l a b e l i n g of a n y of t h e H i n d II + I I I fragm e n t s . I n t h e e v e n t u a l l y of a c t u a l r e p l i c a t i o n , a g r a d i e n t w a s to be e x p e c t e d in t h e l a b e l i n g of t h e f r a g m e n t s , s u c h as is o b s e r v e d in v i v a [5, 22]. T h a t t h i s 'was not t h e c a s e suggests t h a t in v i t r o l a b e l i n g of SV4'0 D N A w i t h t h e c y t o p l a s m i c e x t r a c t s r e s u l t s e s s e n t i a l l y f r o m a r a n d o m i n c o r p o r a t i o n of n u c l e o tides into preexisting strands. P a r t of e a c h of t h e r e s t r i c t i o n e n z y m e digests s h o w n in figure 2 w a s n e x t a n a l y z e d f o r d e n s i t y , first at n e u t r a l p H in CsC1, t h e n at a l k a l i n e p H i n Cs2SO 4. T h e CsC1 g r a d i e n t s s h o w e d t h a t t h e in v i t r o l a b e l e d D N A p r o d u c t s in b o t h s a m p l e s h a d t h e s a m e d e n s i t y as t h e D N A t e m p l a t e s u s e d in t h e r e s p e c t i v e assays (not s h o w n ) .

of t h e e x p e r i m e n t ) , a n d of t h e 32p l a b e l e d l i g h t s t r a n d ( a p p r o x i m a t e l y 1.38). As c a n be s e e n in t h e l o w e r p a n e l of t h e figure, t h e d e n s i t y p r o f i l e 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 w i t h E3HJ-dTMP a n d C .P.M ~C

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each of the Hind II 4- III digests shown in the preceding figure was centrifuged in alkaline C~S04 as deseribed under Material and M'ethods (method B). Upper panel : [3H]-dTMP and dGMP labeled DNA synthesized in the assay using 32p labeled light DNA as the template. Lower panels : 3H labeled DNA synthesized in the control assay using .~2p labeled light DNA as the template. The symbols are the same as those in figure 2.

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led DNA's in the fractions of the neutral sucrose gradients from the preceding experiment (brackets in panels A and C, fig. 1 ) w e r e treated with Hind II 4- III restriction enzymes as described under Material and Methods, after which part of the samples was analyzed by electrophoresis in 4 per cent polyaerylamide gels. Upper panel : [3H]-dTMP labeled DNA synthesized in the assay using [14C]-BrdU DNA as template. Lower panel : control assay using 32p labeled light DNA as template. Symbols : A - - A : z4C ; O - - - O :3H ; O - - O : 32p. The letters A-J refer to the fragments as denominated by Nathans and Danna [23].

T h e p a t t e r n s of t h e CszSO 4 g r a d i e n t s are illus t r a t e d in figure 3. T h e t w o v e r t i c a l d o t t e d l i n e s i n d i c a t e t h e a v e r a g e d e n s i t y of t h e I14C~-BrdU l a b e l e d D N A s t r a n d (1.4'0~5 u n d e r t h e c o n d i t i o n s BIOCHIMIE, 1976, 58, n ° 9.

[3H]-dGMP, w a s i d e n t i c a l to t h a t of t h e l i g h t D ~ A s t r a n d s u s e d as t e m p l a t e . In t h e c o n v e r s e e x p e r i m e n t , h o w e v e r , u s i n g ~14C]-BrdU s u b s t i t u t e d D N A t e m p l a t e s ( u p p e r p a n e l in fig. 3), o n l y h a l f of t h e in v i t r o l a b e l e d D N A h a d t h e d e n s i t y of t h e h e a v y template strands, while the other half had the a v e r a g e d e n s i t y of l i g h t DNA. T h i s r e s u l t e d in a s h i f t in t h e d e n s i t y of t h e n e w l y l a b e l e d m a t e r i a l towards lighter densities. Since the template DNA u s e d in t h e e x p e r i m e n t w a s m a d e of o n e h e a v y s t r a n d a n d one l i g h t [31], t h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t most, if n o t all, of t h e d e o x y r i b o n u c l e o t i d e s i n c o r p o r a t e d in v i t r o b y t h e S100 s y s t e m w a s due to t h e r a n d o m i n s e r t i o n of n u c l e o t i d e r e s i d u e s i n t o b o t h s t r a n d s of the template DNA molecules.

R e p a i r s g n t h e s i s of S V 4 0 D N A i n v i t r o . To check this conclusion, the reverse experim e n t was performed, using as a template light SV40 DNA molecules p r e l a b e l e d w i t h ~2p in vivo, the p r o d u c t being labeled w i t h B r d U T P i n lieu of dTTP, together w i t h I3H!-dGTP. This type of in vitro density labeling has been used p r e v i o u s l y to differentiate r e p l i c a t i o n from r e p a i r synthesis [6, 19]. The DNA labeled i n this assay was analyzed 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 n e u t r a l pH as in the p r e c e d i n g experiment. Both the template (labeled w i t h ~ P in vivo) and p r o d u c t DNA molecules (labeled w i t h [3H]-dGTP and B r d U T P in vitro) s e d i m e n t e d as illustrated in the e x p e r i m e n t of figure 1, w i t h the totality of the former and the m a j o r i t y of the latter recovered at more t h a n 16S (not shown). The m a t e r i a l s e d i m e n t i n g at 16-25S was pooled. Analysis of this m a t e r i a l at alkaline pH showed that about 2/3 of both the DNA p r o d u c t a n d of the DNA template molecules s e d i m e n t e d at 53S liike SV4,0 DNA c o m p o n e n t I

ii:i

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Fro. 4. - - Density distribution of the DNA labeled in vitro with BrdUTP + [3H]-dGTP. A standard assay was carried out as described in the legend to figure 1 except that the template was 32p labeled SV40 DNA component I, and that [3H]-dTTP was replaced by BrdUTP (100 i~M). Incubation was for 120 min. at 32°C. The labeled material was processed as described in the text and in the legend to figure 2. Panel A : analysis by polyacrylamide gel electrophoresis of the fragments generated by Hind II + I I I restriction endonucleases. Panel B : CsC1 density gradie'nt centrifugation at neutral pH of the Hind II -~- III digest. The bracket refers to the fractions which were used for analysis at alkaline pH. Panel G : Cs~SO~ density gradienL centrifugation at alkaline pH (according to method A in Materials and Methods) of the material from the fractious indicated by the bracket in panel B. Symbols : ©--© : 32p

; O---O

: 3H.

BIOCH1MIE, 1976, 58, n ° 9.

1117

molecules, whereas the r e m a i n i n g 1/3 s e d i m e n t e d at 16-18S like SV40 DNA c o m p o n e n t II. No small m o l e c u l a r weight fragments were detected. The p r o d u c t labeled in vitro i n the presence of B r d U T P had therefore the same s e d i m e n t a t i o n b e h a v i o r as that labeled d u r i n g a s t a n d a r d incub a t i o n i n the presence of dTTP. The DNA was next treated w i t h the r e s t r i c t i o n enzymes Hind II + I i I as in tile p r e c e d i n g exper i m e n t , and part of the digest was analyzed by electrophoresis on 4 per cent p o l y a c r y l a m i d e gel (fig. 4, panel A). The t r i t i u m label was recovered i n all of the eleven fragments generated by the r e s t r i c t i o n enzymes. The a p p a r e n t d i s c r e p a n c y b e t w e e n ~H and 32p counts in fragments B, C and D, most likely reflects the difference in base composition of the c o r r e s p o n d i n g regions of the DNA [5] since the only t r i t i u m label i n this exper i m e n t was i n dGMP residues. The DNA fragments generated by the Hind rest r i c t i o n enzymes were next analyzed for density at n e u t r a l pH by i s o p y c n i c c e n t r i f u g a t i o n i n CsC1 (fig. 4, p a n e l B). I n spite of the fact that the in vitro i n c u b a t i o n h a d b e e n c a r r i e d out i n the p r e s e n c e of B r d U T P , the great m a j o r i t y of the p r o d u c t DNA, labeled w i t h [3HJ-dGTP d u r i n g the assay (closed circles), had a d e n s i t y i d e n t i c a l to that of the light, ~2p labeled, DNA molecules used as template (open circles). A m i n o r part of the p r o d u c t DNA was recovered at a density slightly greater t h a n that of the template DNA, again i n d i c a t i v e of the difference i n base c o m p o s i t i o n of the v a r i o u s SV40 fragments generated by the Hind r e s t r i c t i o n enzymes. The fractions of the CsC1 gradient sho~vn betw e e n brackets i n p a n e l B of figure 4 w e r e pooled, and the density of the separated s t r a n d s was analyzed by c e n t r i f u g a t i o n in atkaline Cs2SO 4 (fig. 4, p a n e l C). No difference i n d e n s i t y could be detected b e t w e e n the s t r a n d s w h i c h h a d i n c o r p o rated the dGMP a n d BrdUMP residues in vitro a n d those w h i c h h a d i n c o r p o r a t e d the ~2p label in vivo. This e x p e r i m e n t confirms the results of the p r e c e d i n g one, a n d strengthens the c o n c l u s i o n that the in vitro 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 b y the c y t o p l a s m i c extract is due to r a n d o m insertion of d e o x y r i b o n u c l e o t i d e residues into both s t r a n d s of the template DNA molecule, a n d not to the actual r e p l i c a t i o n of the template. Use of the system.

cytoplasmic

extract

as a repair

To f u r t h e r ascertain this conclusion, it was verified w h e t h e r the SIO,O system could be used

L. M a r r y a n d coll.

1118

as a r e p a i r system and w o u l d for e x a m p l e ensure the labeling and closure of SV40 D NA c o m p o nent II molecules. F o r that purpose, 32p l a b e l e d c o m p o n e n t II DNA m o l e c u l e s w e r e p r e p a r e d t h r o u g h l i m i t e d digestion of SV40 DNA c o m p o nent ] w i t h DNase and purified as d e s c r i b e d in Material and Methods. T h e r e s u l t i n g c o m p o n e n t II m o l e c u l e s w e r e used as template, t o g e t h e r w i t h v a r i o u s p r o p o r t i o n of u n l a b e l e d c o m p o n e n t I molecules, in a s t a n d a r d assay in the p r e s e n c e of a $100 extract, and of [3H]-dTTP and [3HI-dGTP to label the p r o d u c t . The 32p labeled DNA comp o n e n t II m o l e c u l e s of the t e m p l a t e did not c o n t a i n any SV40 D:NA c o m p o n e n t I m a t e r i a l , as s h o w n in figure 5 (panel D). C.P.M.

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Fro. 5 .-- Influence of the nalure of the SVSO I)NA lemplate. A cytoplasmic extract was incubated in a standard assay using 50 i~tM each [3HI-dTTP and [3H]-dGTP as precursors. The template was either 5.4 :zg of 32p labeled purified DNA component I1 molecules alone (670 cpm per ~g of DNA), or 3.2 ~tg of :t2p component II molecules added with 3.2 lxg of unlabeled SV40 DNA component I molecules, or 6.4 :vg of unlabeled DNA component I molecules alone (panels A, B and C respectively). The DNA component II molecules were prepared as 4escribed in M~ethods. The reaction was stopped after 160 min. of incubation at 32,°C and the labeled molecules were extracted with phenolchloroform, precipitated with ethanol, and analyzed by alkaline sucrose gradient centrifugation. Un:incubated 32p DNA component II molecules were sedimented in parallel (panel D). Symbols : O--O : 32p ; ® - - - 0 : 3H.

In the r e a c t i o n using only c o m p o n e n t II molecules as t e m p l a t e (fig. 5, p a n e l A), 76 p e r cent of the 32p labeled c o m p o n e n t II t e m p l a t e m o l e c u l e s w e r e c o n v e r t e d to c o m p o n e n t I m o l e c u l e s in the c o u r s e of the assay. D u r i n g t h e same time, t r i t i u m

BIOCHIMIE, 1976, 58, n o 9.

label w a s i n c o r p o r a t e d in vitro into b o t h 53S DNA c o m p o n e n t I and 18S c o m p o n e n t II species. In the r e a c t i o n using 50 p e r cent c o m p o n e n t I and 5,0 p e r cent c o m p o n e n t II m o l e c u l e s as template (panel B) 76 p e r cent of t h e 32p l a b e l e d comp o n e n t II t e m p l a t e m o l e c u l e s w e r e again c o n v e r t e d to c o m p o n e n t I m o l e c u l e s d u r i n g the course of t h e i n c u b a t i o n , and t r i t i u m label was again i n c o r p o r a t e d in vitro into 53S c o m p o n e n t I a n d 18S c o m p o n e n t II species, to t h e same extent and in the same p r o p o r t i o n as in the p r e c e d i n g sample. Finally, in the only p r e s e n c e of DNA component I as t e m p l a t e (panel C), t r i t i u m label w a s also i n c o r p o r a t e d into DNA c o m p o n e n t I and c o m p o n e n t II molecules, in a p p r o x i m a t e l y the same p r o p o r t i o n s as in t h e p r e c e d i n g samples, a l t h o u g h to a s o m e w h a t lesser extent. T h e ~2p labeled DNA c o m p o n e n t II m o l e c u l e s w h i c h h a d been c o n v e r t e d into c o m p o n e n t I by the S100 system in vitro w e r e a n a l y z e d for superh e l i c i t y by CsCt g r a d i e n t c e n t r i f u g a t i o n 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 . The density of DNA c o m p o n e n t II m o l e c u l e s was 1.540, w h i l e that of a u t h e n t i c DNA c o m p o n e n t I is 1.,585. After t h e i n c u b a t i o n p e r i o d , 75 p e r cent of the 32p l a b e l e d DNA w a s r e c o v e r e d in m a t e r i a l w i t h a d e n s i t y of 1.597 (not s h o w n ) . T h e 53S DNA f o r m e d in vitro h a d t h e r e f o r e a density h e a v i e r t h a n that of DNA f o r m I, s h o w i n g that it was m a d e of c o v a l e n t l y closed c i r c u l a r m o l e c u l e s l a c k i n g s u p e r h e l i c i t y (relaxed c o m p o n e n t I). This result confirms the results r e p o r t e d in the p r e c e d i n g p a p e r [9], indic a t i n g that t h e DNA p r o d u c t labeled in vitro by a c y t o p l a s m i c system is r e l a x e d c o m p o n e n t I molecules. It is also consistent w i t h the fact that DNA f o r m II m o l e c u l e s i n c u b a t e d in the p r e s e n c e of purified ligase are c o n v e r t e d into r e l a x e d circles, as s h o w n by others [1, 17, 261.

Influence of histones. It a p p e a r s f r o m t h e above e x p e r i m e n t s that the c y t o p l a s m i c system only s u p p o r t s in vitro r e p a i r synthesis of SV40 DNA. Since, h o w e v e r , all of the p r e c e d i n g e x p e r i m e n t s w e r e p e r f o r m e d using n~ked SV40, DNA, it was asked w h e t h e r the same w o u l d be t r u e of h i s t o n e - b o u n d DNA. T h e r e is a m p l e e v i d e n c e that r e p l i c a t i n g DNA molecules are t i g h t l y associated w i t h histones i n s i d e the i n f e c t e d cell n u c l e u s [3, 7, 11-15, 24, 25, 28]. In o r d e r to c h e c k the influence of histones on the specificity of the system, SV40 n u c l e o h i s t o n e s ( m i n i c h r o m o s o m e s ) w e r e used as t e m p l a t e in lieu of n a k e d DNA. T h e m i n i c h r o m o s o m e s w e r e e i t h e r r e c o n s t i t u t e d in vitro, a c c o r d i n g to Oudet et al.

Repair synthesis of SV40 DNA in vitro. [24], or e x t r a c t e d f r o m i n f e c t e d ceils, a c c o r d i n g to Green et al [12]. As seen in figure 6, the efficiency of reconstituted m i n i c h r o m o s o m e s as t e m p l a t e was only 1015 p e r cent that of naked DNA (on a mg of DNA

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d u r i n g the i n c u b a t i o n at 32 °, r e s u l t i n g in a m o r e p r o n o u n c e d f r a g m e n t a t i o n of both the DNA template and of the DNA p r o d u c t m o l e c u l e s d u r i n g the assay. In no i n s t a n c e d u r i n g the course of these exper i m e n t s w e r e w e able to detect m a t e r i a l sedim e n t i n g as R.I. m o l e c u l e s I18, 27]. This shows that the p r e s e n c e of b o u n d histones on the DNA t e m p l a t e molecules, t h o u g h i n h i b i t o r y t o w a r d the r e p a i r a c t i v i t y of the c y t o p l a s m i c extract, was not sufficient to p r o m o t e r e p l i c a t i o n . T h e h i g h m o l e c u l a r w e i g h t DNA p r o d u c t labeled in vitro in the p r e c e d i n g e x p e r i m e n t s w a s digested by t h e Hind II + III r e s t r i c t i o n e n d o n u c l e a s e s and the r e s u l t i n g f r a g m e n t s w e r e a n a l y z e d by e l e c t r o p h o r e s i s in p o l y a c r y l a m i d e gels. No prefer e n t i a l l a b e l i n g of any of the Hind II + I I I fragments w a s d e t e c t e d (not shown). It is clear f r o m these results that t h e use of v i r a l c h r o m a t i n in lieu of v i r a l DNA did not i m p a r t to the c y t o p l a s m i c e x t r a c t the ability to r e p l i c a t e v i r a l DNA.

Specificity of the reaction. i'o

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Fro. 6. - - Template activity of viral nucleoproteins reconstituted in vitro. The template .activity of reconstituted viral nucloprotcins (panel A) was compared to that of viral DNA component I molecules (panel B)). The same SV40 DNA molecules labeled with 32p were used as a template (open circles in both panels), either as such or as a reconstituted nucleoprotein complex after incubation with histoncs (see Material and Methods). Incubation with cytoplasmic extract in a complete system was for 60 min. at 32°C. The material labeled in vitro was analyzed by sucrose gradient ccntrifugation at neutral pH. Symbols : C--O : 32p ; @---@ : [3H]-dTMP and -dGMP incorporated.

basis), as j u d g e d f r o m tile extent of i n c o r p o r a t i o n of n u c l e o t i d e s into DNA c o m p o n e n t I and component II m o l e c u l e s in vitro. This suggests that h i s t o n e - c o a t e d DNA is a p o o r t e m p l a t e for DNAp o l y m e r a s e s . Also, the use of m i n i c h r o m o s o m e s in lieu of n a k e d DNA resulted in the f o r m a t i o n of a large a m o u n t of small m o l e c u l a r w e i g h t fragments. S i m i l a r results w e r e o b t a i n e d w i t h n a t u r a l m i n i c h r o m o s o m e s o b t a i n e d by T r i t o n X 100 e x t r a c t i o n [12] f r o m i n f e c t e d cell n u c l e i (not shown). T h e large amount of small m o l e c u l a r w e i g h t f r a g m e n t s f o u n d in both e x p e r i m e n t s suggests that t h e DNA in the c h r o m a t i n was less available to the action of the DNA ligase in the SI'00 e x t r a c t than n a k e d DN,A. Alternatively, it could be that the histones w e r e n a t u r a l l y contam i n a t e d by nucleases, w h i c h w e r e a c t i v a t e d

BIOCHIMIE, 1 9 7 6 ,

58, n o 9.

T h e question ~¢e asked last was w h e t h e r the a c t i v i t y of the c y t o p l a s m i c e x t r a c t was specifically r e l a t e d to SV40 i n f e c t i o n or not. It is k n o w n that the r e p l i c a t i o n of SV40 DNA in vlvo r e q u i r e s the p a r t i c i p a t i o n of an early v i r a l gene p r o d u c t , w h i c h seems to act as an i n i t i a t o r for each r o u n d of v i r a l DNA synthesis [2, 8, 20, 29, 30]. It follows that specific in vivo r e p l i c a t i o n of SV40 DNA should only be feasible w i t h extracts f r o m SV40 i n f e c t e d ceils. F u r t h e r m o r e , the a c t i v i t y of extracts prep a r e d f r o m ceils i n f e c t e d w i t h the tsA3'0 m u t a n t of SV40 s h o u l d display t h e r m o s e n s i b i l i t y in vitro, since the t h e r m o s e n s i t i v i t y of this m u t a n t resides in its i n i t i a t i o n f u n c t i o n in vivo [8, 29]. A c c o r d i n g l y , c y t o p l a s m i c e x t r a c t s w e r e prep a r e d f r o m e i t h e r u n i n f e c t e d ceils, or f r o m cells i n f e c t e d w i t h the tsA30 mutant, and t h e i r activity w a s c o m p a r e d to that of extracts f r o m w t SV40 i n f e c t e d cells. As s h o w n by figure 7 (center panels) no differ e n c e w a s d e t e c t e d in the n a t u r e of the DNA m a d e in vitro by extracts f r o m i n f e c t e d or u n i n f e c t e d cells, except that the i n f e c t e d cell e x t r a c t (upper nanels) w a s s o m e w h a t m o r e active at m a k i n g comp o n e n t I DNA m o l e c u l e s in vitro, on a mg of protein basis, t h a n the u n i n f e c t e d cell e x t r a c t (lower panels). It is not c l e a r w h e t h e r this is or not r e l a t e d to the i n c r e a s e d f r a g i l i t y of the cell nucleus in the SV4'0 i n f e c t e d cells [10], w h i c h could entail g r e a t e r leakage of n u c l e a r enzymes into the c y t o p l a s m i c f r a c t i o n at the t i m e w h e n the extracts

L. M a r t g a n d coll.

1120

w e r e p r e p a r e d . In a n y case, it a p p e a r s e x t r a c t s f r o m u n i n f e c t e d cells w e r e p e r f e c t l y to i n c o r p o r a t e l a b e l e d n u c l e o t i d e s i n t o D N A p o n e n t I a n d c o m p o n e n t II m o l e c u l e s in ( p a n e l E). H ~0

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T h e l a c k of s p e c i f i c i t y of t h e c y t o p l a s m i c s y s t e m w a s also d e m o n s t r a t e d u s i n g e x t r a c t s f r o m tsA30 i n f e c t e d cells a n d c o m p a r i n g t h e i r a c t i v i t y at 32, 37 a n d 42°C. T h e h i g h e r t h e t e m p e r a t u r e , t h e m o r e i n c o r p o r a t i o n of t h e f o u r d N T P ' s i n t o v i r a l D N A w a s s t i m u l a t e d . I n no i n s t a n c e w e r e w e a b l e to o b s e r v e a d i f f e r e n c e b e t w e e n t h e i n f l u e n c e of t e m p e r a t u r e on t h e a c t i v i t y of t h e tsA30 i n f e c t e d cells e x t r a c t , a n d on t h a t of a w t SV4~0 i n f e c t e d cells extract. DISCUSSION.

/

FIG. 7. Requirements for the in vitro labelling of SV$O DNA using extracts from SV40 infected and uninfected cells. SV49 DNA component I molecules (40 l~g/ ml) were incubated with cytoplasmic extracts from either SV40 infected cells (top panels) or uninfected cells (bottom panels) at 2.14 and 1.94 mg of protein per ml respectively. Incubation was in 20 mH Hepes pH 7.8, 5 mM MgCI~, 1 mM MnC12, 100' !lzg/ml BSA, 100 ~tM each dGTP, dCTP, and dA~ofP, 50 i~M [3H]-dTTP (1000 cpm/pmole), for 1 hr at 32 C. The material was then analyzed by sucrose gradient eentrifngation at ~H 13. Panels A and D' : incubation in the presence of mM ATP ; panels B and E : incubation in the presence of 1 mM ATP supplemented with 6 mM PEP and 5 ~ttg of pyruvate kinase per ml ; panels C and F : same as pr.eeeding but in t,he additi, onal presence of 100 ~M each of GTP, CTP and UTP. The arrows refer to the position of 32p labeled DNA marker. -

as t h o s e f r o m SV40 i n f e c t e d cells at i n c o r p o r a t i n g l a b e l e d t r i p h o s p h a t e s i n t o SV40 c o m p o n e n t I a n d c o m p o n e n t II D N A m o l e c u l e s in vitro (not s h o w n ) .

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that able comvitro

-

T h e s y n t h e s i s of u n i t l e n g t h SV40 D N A m o l e cules u s i n g e i t h e r i n f e c t e d o r u n i n f e c t e d c e l l e x t r a c t s r e q u i r e d t h e p r e s e n c e of an e n e r g y g e n e r a t i n g s y s t e m in a d d i t i o n to t h a t of A T P ( c o m p a r e p a n e l s A a n d D to p a n e l s B a n d E r e s p e c t i v e l y ) , w h i c h suggests t h a t b o t h e x t r a c t s w e r e c o n t a m i n a t e d w i t h A T P a s e s . A T P c o u l d n o t be r e p l a c e d b y GTP, C T P o r U T P (not s h o w n ) , n o r did t h e p r e s e n c e of GTP, C T P a n d U T P in a d d i t i o n to t h a t of A T P h a v e a n y effect on t h e n a t u r e o r t h e e x t e n t of t h e l a b e l i n g in vitro ( c o m p a r e r i g h t a n d m i d d l e p a n e l s in figure 7). Since the preceding experiment had been perf o r m e d in a i n c u b a t i o n assay l a c k i n g salt a n d tRNA, w h i c h w e s h o w e d in t h e p r e c e d i n g p a p e r to m i n i m i z e t h e i n f l u e n c e of t h e e n d o n u c l e a s e s in t h e e x t r a c t [9], t h e e x p e r i m e n t w a s r e p e a t e d u s i n g m o r e s t r i n g e n t c o n d i t i o n s . E v e n in t h e p r e s e n c e of t R N A a n d KCI, h o w e v e r , t h e e x t r a c t s f r o m u n i n f e c t e d cells w e r e f o u n d to be a h n o s t as a c t i v e

BIOCHIMIE, 1976, 58, n ° 9.

The present experiments confirm and extend o u r p r e v i o u s o b s e r v a t i o n s [9] t h a t s o l u b l e c y t o p l a s m i c e x t r a c t s f r o m m o n k e y cells are able to utilize SV4'0 D N A c o m p o n e n t I m o l e c u l e s as a t e m p l a t e f o r t h e in vitro i n c o r p o r a t i o n of d e o x y r i b o nucleoside triphosphates into DNA molecules which have the same sedimentation character i s t i c s as a u t h e n t i c v i r a l c o m p o n e n t II a n d relaxed component I DNA molecules. S u c h an i n c o r p o r a t i o n does not s e e m h o w e v e r to r e s u l t f r o m t h e o r d e r e d r e p l i c a t i o n of t h e v i r a l g e n o m e in vitro, but r a t h e r f r o m t h e r a n d o m i n c o r p o r a t i o n of n u c l e o t i d e s i n t o p r e e x i s t i n g SV40 D N A m o l e c u l e s . I n t h a t sense, it is r e m i n i s c e n t of a D N A r e p a i r r e a c t i o n . T h i s c o n c l u s i o n is b a s e d on t h e fact t h a t t h e d e n s i t y l a b e l i n g of SV40 D N A u s i n g l i g h t v i r a l D N A as a t e m p l a t e in t h e p r e s e n c e of B r d U T P , d i d n o t r e s u l t in an i n c r e a s e in t h e d e n s i t y of t h e D N A p r o d u c t l a b e l e d in vitro. C o n v e r s e l y , t h e use of B r d U s u b s t i t u t e d (heavy) SV40 D N A ~s a t e m p l a t e in a r e a c t i o n u s i n g [SH~d T T P a n d [ 3 H ] - d G T P to l a b e l t h e p r o d u c t in vitro, did not r e s u l t in a d e c r e a s e in t h e d e n s i t y of t h e D N A o r o d u c t . T h i s s h o w s t h a t t h e in vitro i n c o r p o r a t i o n w a s t h e r e s u l t of b r e a k d o w n a n d r e s y n t h e s i s of t h e t e m p l a t e D N A m o l e c u l e s , a n d not of net s y n t h e s i s of n e w D N A s t r a n d s . S u c h a c o n c l u s i o n w a s also s t r e n g t h e n e d by t h e fact t h a t t h e e l e v e n D N A f r a g m e n t s g e n e r a t e d b y t h e r e s t r i c t i o n e n d o n u c l e a s e s f r o m Hemophilus influenzae -were e q u a l l y l a b e l e d in all of t h e newly labeled DNA products that we examined i r r e s p e c t i v e of t h e d u r a t i o n of t h e in vitro i n c u bation. M o r e o v e r , w e d i d not o b s e r v e a n y fast s e d i m e n t i n g m a t e r i a l , r e m i n i s c e n t of SV4,0 ILL m o l e c u l e s [18, 277, e v e n t a f t e r t h e s h o r t e s t i n c u b a t i o n t i m e s ( u n p u b l i s h e d results). F i n a l l y , t h e a c t i v i t y of t h e c y t o p l a s m i c f r a c t i o n s f r o m u n i n f e c t e d m o n k e y cells w a s not f o u n d to be signifi-

Repair

synthesis

of SV40

DNA

in vitro.

1121

c a n t l y d i f f e r e n t f r o m t h a t of SV40 i n f e c t e d cells, nor did extracts from tsA30 infected cells exhibit g r e a t e r t h e r m o s e n s i t i v i t y t h a t t h o s e f r o m w t SV40 i n f e c t e d cells.

a n d w h a t r e l a t i o n , if a n y , t h i s in v i t r o s y s t e m h a s w i t h t h e in v i v o r e p a i r s y n t h e s i s of D N A i n t h e l i v i n g cell. W o r k is i n p r o g r e s s a l o n g t h e s e l i n e s .

As a f u r t h e r p r o o f of t h e l a c k of s p e c i f i c i t y of this system, we have recently found that cytoplasmic extracts from HeLa cells (which are not p e r m i s s i v e f o r t h e r e p l i c a t i o n of SV4'0 i n v i v o ) , c a n also b e s u c c e s s f u l l y u s e d i n v i t r o f o r t h e l a b e l i n g of SV40 DNA.

B~SUM~.

Altogether, these results demonstrate that the v i r a l i n i t i a t o r p r o t e i n , w h i c h is t h e p r o d u c t of t h e A g e n e of SV40, a n d w h i c h is r e q u i r e d f o r t h e i n i t i a t i o n of e a c h r o u n d of v i r a l D N A r e p l i c a t i o n in v i v o [2, 8, 20, 29, ~0], is n o t i n v o l v e d i n t h e i n v i t r o a c t i v i t y of t h e c y t o p l a s m i c e x t r a c t . T h e s a m e w a s f o u n d t o b e t r u e of t h e c y t o p l a s m i c s y s t e m s u p p l e m e n t e d w i t h n u c l e a r e x t r a c t s (N100). T h e a d d i t i o n of n u c l e a r e x t r a c t s w a s o f t e n i n h i b i t o r y f o r t h e a c t i v i t y of t h e c y t o p l a s m i c f r a c t i o n . I n n o c a s e h o w e v e r d i d it a p p a r e n t l y c o n f e r v i r a l s p e c i ficity to the system. The cytoplasmic system described here and in t h e a c c o m p a n y i n g p a p e r is t h e r e f o r e n o t a p p l i c a b l e t o t h e in v i t r o s t u d y of t h e r e p l i c a t i o n of SV40 DNA. It m i g t h o w e v e r b e of s o m e u s e f o r t h e s t u d y of v i r a l o r c e l l u l a r D N A r e p a i r s y n t h e s i s i n v i t r o . I n d e e d , i n c u b a t i o n of c y t o p l a s m i c e x t r a c t s w i t h SV40 D N A c o m p o n e n t II m o l e c u l e s b e a r i n g s i n g l e s t r a n d b r e a k s , r e s u l t e d i n t h e s e a l i n g of t h e b r e a k s , i.e. i n t h e c o n v e r s i o n of t h e D N A c o m p o n e n t I I m o l e c u l e s to D N A c o m p o n e n t I molecules. In addition, the newly made DNA I molecules were internally labeled through incorp 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 residues from t h e i n c u b a t i o n m e d i u m . T h e p r o p o r t i o n of r e s u l ting DNA component I molecules in the template D N A w a s a l w a y s t h e s a m e , i n d e p e n d e n t of t h e p r o p o r t i o n of D N A c o m p o n e n t I I at t h e s t a r t of incubation. T h i s s u g g e s t s t h a t t h e r e w a s a fine b a l a n c e b e t w e e n c o n t i n u o u s b r e a k d o w n a n d r e p a i r of t h e a d d e d D N A m o l e c u l e s d u r i n g t h e c o u r s e of t h e i n c u b a t i o n . It is l i k e l y t h a i t h e d e g r a d a t i o n , l a b e l i n g a n d r e p a i r of t h e t e m p l a t e D N A w a s i n i tiated by the single strand cleavage of a DNA m o l e c u l e u n d e r t h e a c t i o n of a n e n d o n u c l e a s e . T h i s w o u l d b e f o l l o w e d b y p o s s i b l e e x t e n s i o n of t h e g a p t h r o u g h t h e a c t i o n of e x o n u c l e a s e ( s ) , r e p a i r s y n t h e s i s b y a D N A p o l y m e r a s e , a n d clos u r e of t h e m o l e c u l e b y t h e cell D N A ligase. I t w i l l b e of g r e a t i n t e r e s t to k n o w w h i c h e n z y m e s , specially which endonucle~se(s) and DNA polymerase(s), are involved in that repair synthesis ; BIOCHIMIE, 1976, 58, n ° 9.

On a montr~ que des e x t r a i t s de cellules CV1 infeet~s p a r le SV40 rSpondent h l ' a d d i t i o n e x p 6 r i m e n t a l e de DNA de SV40 p a r l ' i n c o r p o r a t i o u in vitro des 4 d6soxyribonucl6otides dans du DNA v i r a l (Girard et al, Biochimie, ce volume). P o u r d ~ t e r m i n e r si ce syst~r~e assure ou n o n une r~plication r~elle de la matrice DNA, nous avons fait appel h la t e c h n i q u e du m a r q u a g e de densit~ du DNA p a r emploi de BrdU. Dans une p r e m i e r e expSrience, on a utilis~ comme matriee du DNA de SV40 alourdi p a r i n c o r p o r a t i o n de BrdU [14C] in vivo, et on l'a incub~ in vitro en presence de dTTP E3H] et dGTP [3H]. Dans une deuxi~me expdrience, on a utilis6 cornme matrice du DNA tie SV40 marqu6 a.u [32p] in vivo, ct on l'a incub6 in vitro en prdsence de BrdUTP et de dGTP [3H]. Los prod uits form6s au coors des deux exp6riences ont ensuite ~t~ tiig6r~s p a r t r a i t e m e n t aux enzymes de r e s t r i c t i o n Hind II d- III d'Hemophilus influenzae, puts on a d~termin6 leur densit~ en les c e n t r i f u g e a n t h l'6quilibre e n CsC1 h pH n e u t r e ou en (Cs)2SO4 h pH alcalin. Ces exp6ri.ences m o n t r e n t que la densit~ des moldcules m a r q u e e s au cours de l ' i n c u b a tion in vitro est t o u j o u r s la m~me que celle des moldcul,es matrices ut:ilis~es dans la r6,action. Ceci sugg~re qu.e ]e syst~me assure seulemen,t une synth~se de type r6paration, et n o n pas de type r~plication. L'activit~ de r ~ p a r a t i o n semble d~pourvue de sp~cificit~ vira]e, ear ]es e x t r a i t s des eeIIules CV~ non infectSes sont presque aussi actifs que ceux des cellules infect~es. L ' i n c u b a t i o n de mol6cules de DNA viral forme II (circulaires ouvertes) avec l ' c x t r a i t cytoplasmique, a b o u t i t ~ la conversion de 75 p. cent d'entre elles en molecules de forme I (cercles covalents) d6pourvues de superh~licit~. L ' u t i l i s a t i o n de DNA viral recouvert d ' h i s t o n e s ne modifie pas s e n s i b l e m e n t les propri6tds du syst~me ~tudi6. Acknowledgmenls. We t h a n k Roger Monier a n d Pierre Nardeux for t h e i r gift of restriction endonuclease f r o m H e m o p h i l u s influenzae, P. Sautibres for the gift of calf t h y m u s histones, and P. C h a m b o n for advice regarding the reconstruction of v i r a l m i n i c h r o m o s o m e s . This work was supported by the D61dgation G6n6rale h la Recherche Scientifique et Technique. REFERENCES. 1. Champoux, J. J. & Dulbecco, R. (1972) Proc. Nat. Acad. Sci. U.S., 69, 145-146. 2. Chou, J. Y., Avi]a, J. a Martin, R. G. (1974) J. Virol., I4, 116-124. 3. Cremisi, C., Pignatti, P. F., Croissant, O. & Yaniv, M. (1976) J. ViroL, 17, 204-211. 4. Danna, K. J. ~, Nathans, D. (1971) Proc. Nat. Acad. Sci. U.S., 68, 2913-2917. 5. Danna, K. J. a Nathans, D. (1972) Proc. Nat. Acad. Sci. U.S., 69, 3097-3100. 6. Depamphilis, M. L., Beard, P. ~ Berg., P. (1975) J. Biol. Chem., 250, 4340-4347. 7. Germond, J. E., Hirt, B., Oudet, P., Gross-Bcllard, M. ,~ C h a m b o n P. (1975) Prof. Nat. Acad. Sci. U. S , 72, 1843-1847.

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