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A system of DNA replication in HeLa nuclei treated with inhibitors of protein synthesis
316
Biochimica et Biophysica Acta, 653 (1981) 316--330 Elsevier/North-Holland Biomedical Press
BBA 99853 A SYSTEM OF DNA R E P L I C A T I O N IN HeLa NUCLEI T R E A T E D WITH INHIBITORS OF P R O T E I N SYNTHESIS
KYOSUKE NAGATA, TAKEMI ENOMOTO and MASA-ATSU YAMADA Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 (Japan) (Received January 19th, 1981 ) Key words: DNA synthesis; Cycloheximide; Puromycin; Cell cycle; (HeLa cell)
Summary An in vitro DNA synthesizing system consisting of isolated nuclei from HeLa cells which had been treated with inhibitors of protein synthesis was investigated. T r e a t m e n t with b o t h 30 pg/ml cycloheximide and 10 pg/ml p u r o m y c i n of S-phase cells reduced the rate of DNA synthesis immediately; however, the overall DNA synthesis c o n t i n u e d for up to 4 h with a diminished rate and then ceased. In the nuclei which were isolated from the cells which had been incubated with these drugs for 6 h, little i ncorporat i on of [3H]TTP into acidinsoluble materials was observed. Addition o f cytosol prepared from cells actively synthesizing DNA induced t h e in co r p o r atio n of [3H]TTP in these nuclei, while little induction was observed by the addition of cytosol prepared from drug-treated cells in spite of the fact th at the latter cytosol stimulated DNA synthesis in isolated nuclei f r o m n o n - tr eated cells. T he induced DNA synthesis was shown to require Mg 2+, all four deoxyribonucleoside triphosphates and ATP, and to proceed discontinuously. The activity inducing DNA synthesis in drug-treated nuclei fluctuated with the phases in a cell cycle and it was n o t ascribed solely to DNA polymerase a nor to DNA ligase.
Introduction DNA replication in mammalian cells is a com pl ex process t hat requires multiple approaches for the elucidation of its molecular mechanism. Up to the preAbbreviations: SDS, sodium dodecyl sulfate; SSC, standard saline/citrate solution (0.15 M NaC1/0.15M sodium citrate); AraCTP, cytosine-~-D-arabinofuranoside-5' triphosphate; BrdUrd, 5-bromo-2'-deoxyuridine; FdUrd, 5-fluoro-2'-deoxyuridine;dThd, thymidine;ddTTP,dideoxy 5'-triphosphate. 0005-2787/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
317 sent, two approaches have been used to study DNA synthesis in mammalian cells. One is in vitro study with purified enzymes. Such an approach should be important to ascertain the properties of individual components of DNA replication machinery, but it is not per se sufficient for understanding DNA replication occurring in intact cells. The other approach is to utilize a subcellular in vitro DNA-synthesizing system, involving cell lysates, isolated nuclei [1--11] and permeable cells [12,13]. These systems support intact replication machineries with endogenous template, enzymes and factors and are able to synthesis DNA in vitro with in vivo fidelity. They have been applied to the study on the mechanisms of DNA replication, such as discontinuous replication [6,7], R N A priming synthesis [14,15] and so on. In addition, these in vitro systems could be used for the identification and the characterization of protein factors participating in the processes of DNA replication because of their advantage of presenting no significant barrier to exogenously added macromolecules. However, little is known a b o u t the factors as yet except for so-called 'cytoplasmic factor(s)', which stimulate DNA synthesis in isolated nuclei by accelerating the formation and ligation of primary short fragments {Okazaki pieces) [4,7--9,16,17]. It has been shown from experiments with various organisms that there exists a close coupling between DNA replication and protein synthesis. Studies with bacteria have shown that protein synthesis is a prerequisite for the initiation of a new cycle of DNA replication b u t is not required for the continuation [18-20]. In mammalian cells, it is observed that inhibition of protein synthesis causes a rapid decrease in DNA synthesis before a replication cycle is completed. The rapid decrease is suggested to be due to the inhibition of the initiation of new replicon [21--23] and the decrease in the DNA chain elongation rate at the replicon already in progress [24--27]. Therefore, it seems likely that newly synthesized proteins, which are turned over rapidly, are required for the above processes. Some of the major advances in understanding the roles of factors in DNA replication of prokaryotic cells came from the works using complementation tests of extracts from m u t a n t cells deficient in the factors to be examined. Unlike prokaryotes, DNA replication mutants of eukaryotic cells are n o t available yet, with the exception of yeast cell division cycle mutants. In order to overcome the above limitiation, we have a t t e m p t e d to establish an in vitro system corresponding to mutants. In this communication we will report complementation of the nuclei which are isolated from the cells treated in vivo with inhibitors of protein synthesis and are deficient in some protein factors required for DNA synthesis, with cytosol which is prepared from cells actively synthesizing DNA. It has been suggested that the cytosol prepared from cells actively synthesizing DNA contains the factor{s) which is different from 'cytoplasmic factor', being detectable only b y use of the nuclei isolated from the cells treated in vivo with inhibitors of protein synthesis. Materials and Methods Cell culture and synchronization. HeLa $3 cells were maintained in monolayer culture in Eagle's minimum essential medium supplemented with 10% calf
318 serum (growth medium) at 37°C. Synchronization was accomplished by the addition of 1 mM h y d r o x y u r e a for 16 h to accumulate the cells at the G1/S boundary. Cells were washed twice with Mgz÷, Ca2÷-free phosphate-buffered saline to release the block and were incubated in the fresh growth medium. Treatment with inhibitors of protein synthesis. At 30 min incubation after the release from the h y d r o x y u r e a block, puromycin and cycloheximide were added at a final concentration of 10 and 30 t~g/ml, respectively, and cells were further incubated for 6 h. Cells were harvested as described in the following section except that trypsinization was carried out in the presence of inhibitors of protein synthesis. Preparation of cell lysate, nuclei, cytoplasm and cytosol. Cells were washed once with Mg 2÷, Ca2÷-free phosphate-buffered saline and 0.05% trypsin containing 0.02% EDTA, successively, and trypsinized with the same solution. The trypsinization was stopped by the addition of cold growth medium and cells were collected by low-speed centrifugation. Collected cells were washed twice with Mg z÷, Ca2÷-free phosphate-buffered saline and once with hypotonic medium containing 10 mM Tris-HC1, pH 7.5 and 2 mM MgCl~ and were stored at --20 or --80°C until use. After thawing cells, cell lysate, nuclei, cytoplasm and cytosol were prepared as described in previous papers [28,29]. Nuclei and cytosol prepared from cells treated with inhibitors of protein synthesis as described above were designated 'drug-treated nuclei' and 'drug-treated cytosol', respectively, and those prepared from cells harvested at 3 h after release from h y d r o x y u r e a block were designated 'S-3 h nuclei' and 'S-3 h cytosol', respectively. Preparation of nuclear salt extract. Nuclei were isolated as described with the exception that the media used contained no NaC1. Isolated nuclei were resuspended in buffer A (60 mM Tris-HC1, pH 8.5/11 mM glucose/0.05% Triton X-100/0.5 mM EDTA/1 mM 2-mercaptoethanol/1 mM MgC12) containing various concentrations of NaC1. After keeping for 60 min at 0°C, the nuclear suspension was centrifuged for 10 min at 2500 rev./min. The supernatant was designated 'nuclear salt extract'. The nuclear salt extract was used after dialysis against buffer A containing 80 mM NaC1. Labeling o f DNA in vivo. Cells were incubated with growth medium containing 0.5 pCi/ml [3H]dThd (70 Ci/mmol) for the indicated time. In the experiments requiring highly labeled DNA, cells were labeled with 10 pCi/ml [3H]dThd in Eagle's minimum essential medium supplemented with 10% dialyzed calf serum in the presence of both 1 • 10 -6 M amethopterin and 5 • 10 -s M adenosine or 5 • 10 -6 M FdUrd to decrease the endogenous thymidine pool. Assay of DNA synthesis in vivo. NaN3 was added at a final concentration of 50 pg/ml to the cells which were labeled with [3H]dThd. Cells were harvested as described above and suspended in Mg 2÷, Ca2+-free phosphate-buffered saline at a concentration of (6--10) • l 0 s cells/ml. 0.5 ml of 0.2% SDS was added to 0.5 ml of the cell suspension to lyse cells. An equal volume of 10% trichloroacetic acid/0.1 M sodium pyrophosphate was added to the lysate. The radioactivity incorporated into 5% trichloroacetic acid-precipitable materials was collected on Whatman GF/C glass-fiber filter, washed with 5% trichloroacetic acid five times and with ethanol and acetone once, successively. After drying the filter, the radioactivity was counted in a toluene-based scintillation solution
319 with a liquid scintillation spectrometer.
Assay o f DNA synthesis in in vitro nuclear system and DNA polymerase activity. The standard assay mixture with a total volume of 0.15 ml contained 50 mM Tris-HC1, pH 8.5, 4 0 m M NaC1, 5 mM MgC12, 5 mM 2-mercaptoethanol, 0.25 mM EDTA, 0.025% Triton X-100, 5.5 mM glucose, 3.3 mM ATP, 33 pM each dATP, dCTP, dGTP, 3.3 pM [3H]TTP (1 Ci/mmol) and 5 • 10 s nuclei with or without cytosol. The reaction was stopped by the addition of 0.85 ml of cold water and 1 ml of 10% trichloroacetic acid. The radioactivity was measured as above. DNA polymerase activity was measured as described previously [28].
Alkaline sucrose gradient centrifugation Cells. Cells were lysed with 4/3 vol. of SDS solution containing 0.5% SDS, 0.05 M Tris-HC1, pH 8.0, 0.15 M NaC1 and 0.015 M EDTA, and incubated at 37°C for 8 h with 1/4 vol. of pronase solution containing 10 mg/ml pronase E, 0.05 M Tris-HC1, pH 8.0, 0.15 M NaC1 and 0.015 M EDTA, which had been preheated at 80°C for 10 min and preincubated at 37°C for 2 h to inactivate DNAase. After the addition of NaOH to 0.1 M, samples were kept for 2 h at room temperature, and then layered on the top of 4.8 ml of a 5--20% sucrose gradient containing 0.1 M NaOH, 0.9 M NaC1 and 0.01 M EDTA and centrifuged for 2.5 h at 106 600 X g in a Hitachi RPS-40T2 rotor at 16°C. The fractions were collected from the b o t t o m of the tube. To each fraction, 200 #g of herring sperm DNA and an equal volume of 10% trichloroacetic acid were added. Acid-insoluble radioactivity was collected on the glass-fiber filter. Nuclei. 1 • 104 nuclei were incubated in 0.3 ml of standard reaction mixture except that the specific activity of [3H]TTP was 3 Ci/mmol. After the incubation for various times at 37~C, samples were treated as described above and loaded on 29 ml of 5--20% sucrose gradients containing 0.1 M NaOH 0.9 M NaC1 and 0.01 M EDTA. Centrifugation was carried out in a Hitachi RPS25 rotor at 51 500 X g for 12 h at 16°C.
Neutral CsCl equilibrium density gradient centrifugation Cells. Cells were lysed in 1 ml of 50 ~g/ml proteinase K solution containing 10 mM Tris-HC1, pH 8.0, 10 mM NaC1, 10 mM EDTA and 0.5% SDS, and incubated for 12 h at 37°C. The lysate was dialyzed for 8 h against 0.01 X SSC, pH 7.0. The dialyzate was adjusted to 10 mM Tris-HC1, pH 8.0, to be digested with 1 mg RNAase A at 37°C for 30 min. After sonication with a Bronson sonifier (at 50 W, 5-times 15-s), 0.3 ml of the aliquot was diluted to 3 ml with 0.01 M Tris-HCl, pH 8.0. 4 g of solid CsC1 was dissolved. The sheared DNA was centrifuged for 65 h at 129 000 X g in a Hitachi RPS 40T2 rotor at 20°C. The fractions were collected from the b o t t o m of the tube. Nuclei. 2 • 106 nuclei were incubated in 0.6 ml of standard reaction mixture except that the specific activity of [3H]TTP was 3 Ci/mmol. To the sample, 0.2 ml of buffer K, containing 40 mM Tris-HC1, pH 8.0, 40 mM NaC1 and 2% SDS, and 0.2 ml of 0.25 mg/ml proteinase K, in 10 mM Tris-HC1, pH 8.0, 10 mM NaC1, 10 mM EDTA and 0.5% SDS, were added successively and processed as described above.
320
Materials. [3H]TTP (47--59 Ci/mmol), [3H]dThd (70 Ci/mmol) and [14C]dThd (57 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, U.K.; ATP, dATP, dCTP, dGTP, TTP and proteinase K were from Boehringer Mannheim; adenosine, dThd, amethopterin, h y d r o x y u r e a and AraCTP from Sigma; CsC1 from Merck; RNAase A from Worthington; pronase E from Kaken-Kagaku; Japan; cycloheximide from Wako-Junyaku, Japan; puromycin from Makor Chemical. Results
Inhibition o f DNA synthesis by inhibitors of protein synthesis To examine the effect of inhibitors of protein synthesis on DNA synthesis in vivo, cells were incubated in the presence of varying concentrations of puromycin or cycloheximide for 1 h and then the incorporation of [3H]dThd for 5 min was measured (Fig. 1A and B). The incorporation was inhibited by 90% at a concentration of 30 pg/ml puromycin or 3 pg/ml cycloheximide. Although cycloheximide inhibited more effectively than puromycin, the degree of inhibition increased scarcely with concentrations of cycloheximide higher than 3 pg/ml. Fig. 2 illustrates the rate of DNA synthesis in the cells which were incubated for various periods in the presence of 10 pg/ml cycloheximide and 30 pg/ml puromycin. The rate fell immediately, and within 5 min after the addition of drugs incorporation was reduced by 70%. Thereafter, the degree of inhibition increased gradually up to 1 h and, upon prolonged incubation, a fairly constant rate of incorporation, about 5% of control, was observed. The other combination of 30 pg/ml cycloheximide and 10 pg/ml puromycin gave a similar result. We used hereafter this combination of drugs to inhibit DNA synthesis. Fig. 3 shows the cumulative curve of DNA synthesis in the presence or absence of both drugs. The incorporation of thymidine in drug-treated cells continued at a diminished rate for up to 4 h and no further incorporation was observed after treatment for 6 h. Analysis o f DNA synthesis in the presence o f inhibitors Evidence from previous reports suggests that the inhibition of DNA synthesis by cycloheximide does not result from the alteration of precursor metabolism [24,25,27]. The reduction of DNA synthesis in drug-treated cells, therefore, may be speculated to be due to the reduction of the rate of chain elongation [24--26] and/or the inhibition of initiation of new replicon [21,22]. To obtain more information a b o u t our system, synthesized DNA was analyzed. Cells were pulse-labeled for 2 min with [3H]dThd at the beginning of S phase and chased in the presence of inhibitors. As shown in Fig. 4, it t o o k 3 h for the short chain to elongate to bulk DNA in the presence of inhibitors, while, in control cells, elongation to bulk DNA was accomplished within 0.5--1 h (not shown). Little decrease of total count was observed during the chase in the presence of inhibitors. To examine the effect of inhibitors on the initiation on new replicon, cells were incubated with BrdUrd for 6 h in the presence of inhibitors and then washed to be freed from inhibitors, and then pulse-labeled with [3H]dThd. Synthesized DNA was analyzed by neutral CsC1 density gradient centrifugation.
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Fig. 1. Effect o f inhibitors o f p r o t e i n s y n t h e s i s o n D N A synthesis. 1 • 10 5 cells w e r e i n c u b a t e d for 2 h after release f r o m the h y d r o x y u r e a b l o c k , and further i n c u b a t e d for 1 h in the presence o f v a t t i n g c o n c e n t r a t i o n o f p u r o m y c i n ( A ) or c y c l o h e x i m i d e (B) and then pulse-labeled for 5 rain w i t h 0.5 /~Ci/ml [3H]dThd: Fig. 2. Kinetics of the i n h i b i t i o n o f D N A s y n t h e s i s b y c o m b i n a t i o n o f c y c l o h e x i m i d e and p u r o m y c i n . Cells w e r e i n c u b a t e d for 2 h after release f r o m the h y d r o x y t t r e a b l o c k and further i n c u b a t e d for i n d i c a t e d p e r i o d in the presence o f b o t h 10 p g / m l c y c l o h e x i m i d e and 3 0 p g / m l p u r o m y c i n and t h e n pulse-labeled for 5 rain w i t h [ 3 H ] d T h d . Fig. 3. T i m e course o f c u m u l a t i v e i n c o r p o r a t i o n of [ 3 H ] d T h d . Cells w e r e i n c u b a t e d for 30 rain after release f r o m the h y d r o x y u r e a b l o c k and t h e n t h e y w e r e labeled w i t h 0.5 p C i / m l [ 3 H ] d T h d in the absence ( o ) o r presence ( e ) o f b o t h 3 0 pg/rnl c y c l o h e x i m i d e and 10 p g / m l p u r o m y c i n for time i n d i c a t e d in the figure.
Most of D N A synthesized after 10 min incubation banded at the light density region (Fig. 5A) and a shift to the half heavy density region was observed after 30 min incubation {Fig. 5B). DNA synthesis in vitro in isolated nuclei treated with inhibitors in vivo or in vitro
To examine whether the inhibition of D N A synthesis by the protein synthesis inhibitors resulted from a direct interaction of the drugs with components of the replication machinery or from the inhibition of protein synthesis, D N A synthesis in isolated nuclei treated with the inhibitors in vitro or in vivo was measured. The D N A synthesis in the nuclei isolated from cells actively synthesizing D N A was not inhibited by in vitro treatment of both 30 #g/ml cycloheximide and 10 ~g/ml puromycin, while a reduction in DNA-synthesizing
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Fig. 4. E f f e c t o f i n h i b i t o r s o f p r o t e i n s y n t h e s i s o n D N A c h a i n e l o n g a t i o n . S y n c h r o n i z e d cells w e r e f r e e d f r o m t h e h y d r o x y u r e a b l o c k a n d i m m e d i a t e l y p u l s e - l a b e l e d f o r 2 m i n (~) in t h e a b s e n c e o f inhibitors. A f t e r pulse-labeling, t h e cells w e r e w a s h e d w i t h Mg 2+, Ca2+-free p h o s p h a t e - b u f f e r e d saline a n d c h a s e d w i t h 1 • 10 -S M u n l a b e l e d d T h d for 1 h (~) a n d 3 h ( e ) in t h e p r e s e n c e o f b o t h 3 0 pg/ml c y c l o h e x i m i d e a n d 1 0 pg/ml p u r o m y e i n . D N A s w e r e p r e p a r e d as d e s c r i b e d in Materials a n d M e t h o d s a n d s u b j e c t e d to 5 - - 2 0 % alkaline s u c r o s e g r a d i e n t centrifugation. Sedimentation w a s f r o m r i g h t to l e f t . T h e a r r o w d e n o t e s t h e c o r r e s p o n d i n g p o s i t i o n o f b u l k D N A p r e p a r e d f r o m cells u n i f o r m l y l a b e l e d f o r 24 h. Fig. 5. N e u t r a l CsCI g r a d i e n t analysis o f D N A s y n t h e s i z e d a f t e r release f r o m i n h i b i t i o n of p r o t e i n synthesis. T h e cells p r e - l a b e l e d w i t h [ 14 C ] d T h d w e r e s y n c h r o n i z e d w i t h h y d r o x y u r e a . A f t e r release f r o m t h e b l o c k , cells w e r e i n c u b a t e d in fresh g r o w t h m e d i u m c o n t a i n i n g 5 • 10 -$ M B r d U r d a n d 5 • 10 -6 M F d U r d ( h e a v y m e d i u m ) . 30 rain a f t e r t h e i n c u b a t i o n , c y c l o h e x i m i d e a n d p u r o m y c i n w e r e a d d e d a t c o n c e n t r a t i o n s of 30 a n d 10 p g / m l , r e s p e c t i v e l y . I n c u b a t i o n w a s c o n t i n u e d for a n o t h e r 5.5 h in t h e h e a v y m e d i u m . Cells w e r e w a s h e d w i t h Mg 2+, Ca2+-free p h o s p h a t e - b u f f e r e d saline a n d p u l s e - l a b e l e d w i t h 2 p C i / m l [ 3 H ] d T h d f o r 1 0 r a i n ( A ) o r 3 0 rain (B) in fresh g r o w t h m e d i u m . • 14C p r e - l a b e l e d ; c , 3 H pulse-labeled.
activity in isolated nuclei was noted when nuclei were isolated from the cells treated with inhibitors in vivo (Fig. 6). The in vitro D N A synthesizing activity was not changed till a 30 min treatment of drugs in vivo. Thereafter, it decreased gradually for up to 4 h, and little activity was observed after 6 h treatment.
Complementation of nuclei with cytosol Nuclei deficient in some proteins involved in D N A replication may become an useful tool for identification and evaluation of such proteins. We next performed complementation experiments. Drug-treated nuclei or S-3 h nuclei were complemented with a cytosol prepared from cells actively synthesizing D N A (S-3 h cells). Fig. 7 shows dose response curve of S-3 h cytosol. [3H]TTP incorporation in both nuclei were enhanced by S-3 h cytosol. In S-3 h nuclei, the incorporation was increased with the amount of cytosol up to three cell equivalents and reached a plateau at higher concentrations. In the case of drugtreated nuclei, the incorporation increased proportionally to the amount of S-3 h cytosol up to five cell equivalents. Fig. 8 shows kinetics of the incorporation of [3H]TTP in drug-treated nuclei complemented with three or five cell equivalents of cytosol. The incorporation in complemented nuclei increased
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Fig. 6. I n v i t r o D N A - $ y n t h e s i z i n g a c t i v i t y in n u c l e i i s o l a t e d f r o m cells t r e a t e d w i t h i n h i b i t o r s o f p r o t e i n s y n t h e s i s . Cells s y n c h r o n i z e d w i t h h y d r o x y u r e a w e r e r e l e a s e d f r o m t h e b l o c k a n d i n c u b a t e d f o r 30 r a i n in fresh g r o w t h m e d i u m a n d t h e n p u r o m y c i n a n d c y c l o h e x i m i d e w e r e a d d e d a t c o n c e n t r a t i o n s o f 10 a n d 3 0 # g / m l , r e s p e c t i v e l y . Cells w e r e i n c u b a t e d in t h e p r e s e n c e o f d r u g s for t h e p e r i o d s i n d i c a t e d a n d n u c l e i w e r e i s o l a t e d f r o m t h e cells. I s o l a t e d n u c l e i (1 • 1 0 6 ) w e r e i n c u b a t e d in t h e s t a n d a r d r e a c t i o n m i x t u r e f o r 2 0 rain at 37~C. Fig. 7. E f f e c t o f S-3 h c y t o s o l o n D N A s y n t h e s i s in S-3 h a n d d r u g - t r e a t e d nuclei. S-3 h a n d d r u g - t r e a t e d n u c l e i w e r e i s o l a t e d f r o m S-3 h a n d d r u g - t r e a t e d cells, r e s p e c t i v e l y , as d e s c r i b e d in Materials a n d M e t h o d s . S-3 h c y t o s o l w a s p r e p a r e d f r o m S-3 h c y t o p l a s m as t h e s u p e r n a t a n t b y c e n t r i f u g a t i o n a t 1 0 5 0 0 0 × g f o r 60 rain. 5 • 105 n u c l e i (o, S-3 h nuclei; o, d r u g - t r e a t e d n u c l e i ) w e r e i n c u b a t e d w i t h S-3 h c y t o s o l in a n a m o u n t f r o m 1 t o 5 cell e q u i v a l e n t s in the s t a n d a r d r e a c t i o n m i x t u r e f o r 20 rain a t 3 7 ° C . E a c h v a l u e w a s n o r m a l i z e d as t h e i n c o r p o r a t i o n p e r 1 • 106 nuclei.
with incubation time up to 5--10 min and slightly upon a continued incubation. Little incorporation was observed in non-complemented nuclei. Table I shows complementation of nuclei with drug-treated cytosol as well as that with S-3 h cytosol. It must be noted that drug-treated cytosol, which had no effect on drug-treated nuclei, increased the incorporation in S-3 h nuclei to nearly the same level as S-3 h cytosol.
Characterization o f DNA synthesis in drug-treated nuclei induced by S-3 h cy tosol To characterize DNA synthesis induced in drug-treated nuclei, the requireTABLE I D N A S Y N T H E S I S IN N U C L E I C O M P L E M E N T E D W I T H C Y T O S O L 5 " 105 n u c l e i w e r e i n c u b a t e d f o r 10 rain at 3 7 ° C in t h e a b s e n c e or p r e s e n c e o f d r u g - t r e a t e d c y t o s o l o r S - 3 h c y t o s o l o f 5 cell e q u i v a l e n t s ( 2 . 5 • 106 cells). E a c h v a l u e in t h e t a b l e i n d i c a t e s t h e i n c o r p o r a t i o n / 1 • 106 nuclei. Nuclei
Cytosol
T M P incorporated (pmol/1 • 10 6 nuclei)
Drug-treated Drug-treated Drug-treated S-3 h S-3 h S-3 h
none drug-treated S-3 h none drug-treated S-3 h
0.62 0.32 2.10 1.02 3.33 3.66
324 m e n t of the system and the effect of several inhibitors were investigated. As shown in Table II, the system required all four deoxyribonucleoside triphosphates, Mg 2÷ and ATP. The optimal level of ATP under the conditions tested was around 3--5 mM. Aphidicolin, which is a specific inhibitor for DNA polymerase ~ [30], inhibited DNA synthesis in the complementation system by 75% at 6/~g/ml. DNA synthesis was also sensitive to AraCTP b u t n o t sensitive to ddTTP. Fig. 9 shows alkaline sucrose gradient centrifugation analysis of DNA synthesized in the complementation system. A b o u t 50% of the radioactivity was recovered in short fragments by 1 min pulse-labeling and the rest was distributed widely in longer DNA chains. When pulse-labeled for 5 min, a marked increase of the radioactivity incorporated into longer DNA chains was observed. After 20 min pulse-labeling, almost all the radioactivity appeared in the longer DNA. The short fragments in the peak near the top of the gradient and longer DNA in the broad peak appearing in the two-thirds from the b o t t o m of the gradient were calculated as being about 200 nucleotide long DNA and 40 S DNA, respectively, using nucleosome m o n o m e r DNA and X phase DNA as markers. To check that DNA synthesized initially as short fragments is a precursor to be elongated to longer DNA, pulse-chase experiments were carried o u t (Fig. 10). By 12.5 min chasing, the radioactivity in the peak of short fragments decreased and migrated to the somewhat longer fraction and the majority of the radioactivity distributed in 40 S DNA. After 17.5 min chase, the radioactivity in both peaks of short fragments decreased and almost all the radioactivity was found in 40 S DNA. In order to characterize DNA synthesis in the complementation system further, synthesized DNA was analyzed by neutral CsC1 equilibrium density gradient centrifugation. Nuclei were isolated from cells which were incubated with BrdUrd during the treatment of the inhibitors of protein synthesis and were
TABLE II REQUIREMENTS OF DNA SYNTHESIS IN DRUG-TREATED NUCLEI COMPLEMENTED WITH S-3 h CYTOSOL
T h e c o m p l e t e s y s t e m w a s as d e s c r i b e d in Materials and M e t h o d s . 5 at 3 7 ° C for 2 0 m i n w i t h S - 3 h c y t o s o l o f 4 c e l l e q u i v a l e n t s . o m i s s i o n o f A T P a n d M g 2 + , S - 3 h c y t o s o l w a s u s e d at a m o u n t o f 5 assay m i x t u r e and i n h i b i t o r s w e r e o m i t t e d and a d d e d , r e s p e c t i v e l y , a s bated
Condition
Percent o f complete system
Complete system
100
-- ATP - - M g 2+
4 0
-- dATP -- dGTP
27 18
-- dCTP
14
-- dATP, -- dGTP -- dATP,-dCTP
16 16
-- dCTP,-dGTP + Aphidicolln (0.2/~g/ml) + Aphidicolin (6 / ~ g / m l )
10 86 25
+ AraCTP
19
(1 raM)
• 105 drug-treated nuclei were incuIn t h e case o f the e x p e r i m e n t s w i t h cell equivalents. C o m p o n e n t s o f the g i v e n in the t a b l e .
325 lo I
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Fig. 8. T i m e c o u r s e o f D N A s y n t h e s i s in d r u g - t r e a t e d n u c l e i c o m p l e m e n t e d w i t h S-3 h c y t o s o l . Drugt r e a t e d n u c l e i (5 • 1 0 s ) w e r e i n c u b a t e d at 3 7 ° C for i n d i c a t e d p e r i o d s in the standard r e a c t i o n m i x t u r e in t h e a b s e n c e ( o ) or p r e s e n c e o f S-3 h c y t o s o l o f 3 cell e q u i v a l e n t s (A) and 5 cell e q u i v a l e n t s ( e ) . Fig. 9. A l k a l i n e s u c r o s e s e d i m e n t a t i o n analysis o f D N A s y n t h e s i z e d in in vitro c o m p l e m e n t a t i o n s y s t e m . Cells s y n c h r o n i z e d w i t h h y d r o x y u r e a w e r e r e l e a s e d fTom the b l o c k and i n c u b a t e d for 3 0 rain in fresh g r o w t h m e d i u m and t h e n c y c l o h e x i m i d e and p u r o m y c i n w e r e a d d e d at c o n c e n t r a t i o n s o f 3 0 and 1 0 # g / m l , r e s p e c t i v e l y . N u c l e i w e r e i s o l a t e d f r o m t h e cells ( d r u g - t r e a t e d n u c l e i ) . S-3 h c y t o s o l w a s prepared f r o m cells which were synchronized a n d released into S phase for 3 h. I • I06 drug-treated nuclei were incubated with S-3 h cytosol 5 cell equivalents (5 • 106) for 1 m i n (©), 5 rain (e) and 20 m i n (A) at 3 7 ° C in the standard reaction mixture except that the specific activity of [ 3 H ] T T P w a s 3 Ci/mmol. Sedimentation was from right to left.
labeled with [3H]TTP in vitro. DNA synthesized in non-complemented nuclei banded in the half heavy density region, suggesting that DNA synthesis in the drug-treated nuclei is the continuation of DNA replication at replicons which have been already in progress in vivo (Fig. l l A ) . In the complementation system, synthesized DNA distributed in the peak of half heavy density with a shoulder toward the light density region (Fig. l l B ) . Subcellular distribution of the activity S-3 h cytoplasm was fractionated by centrifugation into several fractions. The complementing activity to drug-treated nuclei was observed both in the cytoplasm and the postmitochondrial fraction (Table III, lines 2 and 4). However, the former fraction incorporated [3H]TTP considerably by itself. This incorporation may be derived from mitochondrial DNA synthesis (Table III, line 3). The activity in the postmitochondrial fraction was further divided into cytosol and microsomal fraction (Table III, lines 5 and 6). Cytosol alone had no capacity to incorporate [3H]TTP into acid-insoluble materials and almost all the complementing activity was recovered in this fraction. Fluctuation of the activity during a cell cycle It has been reported that DNA synthesis in isolated nuclei is stimulated by
326
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:
2
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'
20
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0
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20
30
40
50
F r a c t i o n number
0
60 top
Fig. i0. Alkaline sucrose sedimentation analysis of D N A pulse-labeled and chased in in vitro c o m p l e m e n tation system. Drug-treated nuclei and S-3 h cytosol were prepared as described in the legend to Fig. 9. 1 • 106 drug-treated nuclei were pulse-labeled in the presence of S-3 h cytosol of 5 cell equivalents for 2.5 m i n (o) and chased with a 100-fold excess of unlabeled T T P for 12.5 rain (A) or 17.5 rain (o). Sedimentation was from right to left. Fig. 11. N e u t r a l CsC1 e q u i l i b r i u m d e n s i t y g r a d i e n t analysis of D N A s y n t h e s i z e d in in v i t r o c o m p l e m e n t a t i o n s y s t e m . N u c l e i w e r e i s o l a t e d f r o m t h e cells t r e a t e d as d e s c r i b e d in t h e l e g e n d t o Fig. 5 . 2 • 106 d r u g t r e a t e d n u c l e i w e r e i n c u b a t e d in t h e s t a n d a r d r e a c t i o n m i x t u r e , e x c e p t t h a t t h e specific a c t i v i t y o f [ 3 H ] T T P w a s 3 C i / r a r a o l , f o r 10 rain at 3 7 ° C w i t h o u t ( A ) o r w i t h (B) S-3 h c y t o s o l of 5 cell e q u i v a l e n t s , o, 14C pre-labeled~ o, [ 3 H ] T T P - l a b e l e d .
cytoplasm (cytoplasmic factor), regardless of the phase of cells from which the cytoplasm is prepared [ 1 , 1 1 , 3 1 , 3 2 ] . We have also confirmed this by using S-3 h nuclei and cytoplasms prepared from cells in various phases of a cell cycle. To ascertain whether or not phase-dependency is observed in the case of the
TABLE III S U B C E L L U L A R D I S T R I B U T I O N OF C O M P L E M E N T I N G A C T I V I T Y Cytoplasm, mitochondrial fraction, postraitochondrial fraction, cytosol and raicrosomal fraction were p r e p a r e d f r o m S-3 h cells. M i t o c h o n d r i a l a n d p o s t m i t o c h o n d r i a l f r a c t i o n s w e r e p r e p a r e d f r o m c y t o p l a s m b y c e n t r i f u g a t i o n f o r 2 0 rain at 12 0 0 0 × g. C y t o s o l a n d r a i c r o s o m a l f r a c t i o n w e r e p r e p a r e d f r o m p o s t r a i t o c h o n d r i a l f r a c t i o n b y c e n t r i f u g a t i o n f o r 6 0 rain a t 1 0 5 0 0 0 × g. M i t o c h o n d r i a l a n d r a i c r o s o r a a l fract i o n s w e r e s u s p e n d e d in n u c l e a r i s o l a t i o n b u f f e r . 5 • 105 n u c l e i w e r e i n c u b a t e d f o r 2 0 rain in t h e p r e s e n c e o f 5 cell e q u i v a l e n t s o f e a c h f r a c t i o n . T h e v a l u e in t h e p r e s e n c e o f n u c l e i i n d i c a t e s t h e i n c o r p o r a t i o n p e r 1 • 105 n u c l e i . TMP i n c o r p o r a t e d ( p m o l )
1 2 3 4 5 6
None Cytoplasm Mitochondrial fraction Postraitochondrial fraction Cytosol Microsomal fraction
Fraction only
With d r u g - t r e a t e d n u c l e i
-5.15 4.00 1.49 0.00 0.74
1.27 17.49 6.78 10.42 7.45 2.85
327
complementing activity, cells were synchronized and cytosols were prepared at various phases of a cell cycle. Fig. 12 shows the fluctuation pattern of the activity. The S phase began immediately following release from the hydroxyurea block and lasted for 8--9 h. From 9 to 12 h, the majority of the cells entered into G2 phase and at 15 h the population had almost doubled. The activity was highest in early S phase and decreased gradually with the progression of cell cycle from S to G2 phase. Does the complementing activity arise from a known enzyme? As reported in our previous papers [28,33], most of the DNA polymerase activity in cytosol is derived from DNA polymerase a. After treatment of the inhibitors of protein synthesis for 6 h, the DNA polymerase activity in the cytosol reduced to 60% of the original level. If the complementing activity arises from DNA polymerase a, then a large amount of drug-treated cytosol should restore DNA synthesis in drug-treated nuclei. This was not the case. As indicated in Table I, even five cell equivalents of drug-treated cytosol had no effect on the DNA synthesis in drug-treated nuclei. Furthermore, highly purified DNA polymerase a did not affect the DNA synthesis (not shown). To ascertain that the complementing activity arises from enzymes necessary for the synthesis of high molecular weight DNA, including DNA ligase, we next examined whether or not drug-treated cytosol supports the synthesis of high molecular weight DNA. S-3 h nuclei were incubated with drug-treated cytosol and the product was analyzed by alkaline sucrose gradient centrifugation. In the absence of drug-treated cytosol, little high molecular weight DNA was syn"i 2.5 2C i
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10 bottom
20
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Fig. 12. F l u c t u a t i o n o f c o m p l e m e n t i n g a c t i v i t y during a cell c y c l e . CeLls w e r e s y n c h r o n i z e d w i t h h y d r o x y urea at G 1 / S b o u n d a r y . C y t o s o l s w e r e p r e p a r e d b y h a r v e s t i n g t h e cells at t h e 3-h intervals a f t e r release f r o m t h e b l o c k . T h e a c t i v i t y o f e a c h e y t o s o l w a s a s s a y e d in t h e standard r e a c t i o n m i x t u r e at 37 ° C f o r 2 0 rain b y using d r u g - t r e a t e d n u c l e i . D r u g - t r e a t e d n u c l e i i n c o r p o r a t e d 0 . 4 4 p m o l o f [ 3 H ] T M P b y t h e m s e l v e s . Fig. 1 3 . A l k a l i n e s u c r o s e s e d i m e n t a t i o n analysis o f D N A s y n t h e s i z e d in S-3 h n u c l e i c o m p l e m e n t e d w i t h d r u g - t r e a t e d c y t o s o l . S-3 h n u c l e i and d r u g - t r e a t e d c y t o s o l w e r e p r e p a r e d as d e s c r i b e d in M a t e r i a l s and M e t h o d s . 1 • 1 0 6 S-3 h n u c l e i c o m p l e m e n t e d w i t h d r u g - t r e a t e d c y t o s o l o f 3 cell e q u i v a l e n t s w e r e incub a t e d in t h e standard r e a c t i o n m i x t u r e e x c e p t t h a t t h e s p e c i f i c a c t i v i t y o f [ 3 H ] T T P w a s 3 C i / m m o l , for 1 rain ( o ) and c h a s e d w i t h a 1 0 0 - f o l d e x c e s s o f u n l a b e l e d TTP for 1 0 rain ( o ) . S e d i m e n t a t i o n w a s f r o m right to left.
328 thesized (not shown), while in its presence a marked increase in the synthesis of high molecular weight DNA was observed, and the short fragments synthesized during 1 min pulse-labeling were chased to high molecular weight DNA during another 10 min incubation (Fig. 13).
Does the complementing factor exist in nuclei? S-3 h nuclei were prepared using media containing no NaC1, and nuclear salt extract was prepared as described under Materials and Methods. Addition of the extract to drug-treated nuclei caused no effect on the DNA synthesis, whereas, in the drug-treated homogenate system consisting of drug-treated nuclei and drug-treated cytoplasm, DNA synthesis was restored by the addition of the nuclear salt extract. The activity was extracted efficiently with 80 mM as well as 250 mM NaC1 in the presence of 60 mM Tris-HC1. The combination of 80 mM NaC1 and 60 mM Tris-HC1 is the same condition as that used for the usual preparation of nuclei. Discussion The results presented here show that DNA synthesis in mammalian cells requires concomitant protein synthesis, in agreement with reports by others [21--27,34,35]. The difference between the time course of inhibition of DNA synthesis in vivo (Fig. 2) and in vitro (Fig. 6) may be due to the difference between the systems in that DNA synthesis in isolated nuclei is performed by proteins and enzymes remaining in the nuclei; therefore the situation for DNA synthesis in isolated nuclei, even in non-drug-treated nuclei, is closer to that of drug-treated cells rather than non-drug-treated cells. The behavior of DNA synthesized in the cells freed from inhibitors on neutral CsC1 gradient analysis (Fig. 5) can be explain by the assumption that DNA synthesis restored in the cells occurs predominantly in new replicons and that it takes about 30 min for new DNA chains to elongate in order to be linked with heavy DNA chains which have been synthesized in the presence of inhibitors, or that initiation of new replicons can occur immediately after removal of inhibitors but restoration of DNA synthesis at pre-existing replication forks takes time. Therefore, it seems likely that the combination of cycloheximide and puromycin causes the reduction of both the rate of chain elongation (Fig. 4) and frequency of initiation of new replicon. From the experiments using isolated nuclei, it is unlikely that the reduction of DNA synthesis by the inhibitors is due to the change of metabolism of precursors or to a direct effect of the drugs on replication machinery. Rather, the rapid reduction of DNA synthesis following the addition of the inhibitors is due to the depletion of enzymes a n d / o r proteins which are synthesized in conjunction with DNA synthesis and is turned over rapidly. Therefore, isolated nuclei prepared from the cells treated with the inhibitors may be deficient in some proteins necessary for DNA replication and can be considered as a sort of m u t a n t . By use of such nuclei, it became possible to search for protein(s) required stringently for DNA replication and turned over rapidly, in the cytosol from the cells activity synthesizing DNA. It has been reported that cytoplasm of mammalian cells contains 'cytoplas-
329 mic factor' which stimulates DNA synthesis in isolated nuclei b y accelerating the formation and ligation of primary pieces [4,7--9,16,17]. The possibility that the factor revealed in this study, is n o t identical with 'cytoplasmic factor' is supported by following observations. The level of activity to induce DNA synthesis in drug-treated nuclei fluctuated with phases of a cell cycle, while little fluctuation of the activity of 'cytoplasmic factor' was observed [1,11,31,32]. In addition, although drug-treated cytosol was able to stimulate DNA synthesis in S-3 h nuclei up to nearly the same level as S-3 h cytosol, the activity to induce DNA synthesis in drug-treated nuclei was found in S-3 h cytosol b u t not in drug-treated cytosol. The results shown in Table II suggest that DNA polymerase a is responsible for DNA synthesis in the complementation system. However, the observation, that even high amounts of drug-treated cytosol cannot restore DNA synthesis in drug-treated nuclei (Table I) although the cytosol contains considerable amounts of DNA polymerase ~, suggests that the complementing activity does n o t arise solely from DNA polymerase a. The fact that drug-treated cytosol supports the synthesis of high molecular weight DNA in S-3 h nuclei (Fig. 13) indicates that the cytosol contains a sufficient amount of the enzymes necessary for the step of DNA ligation, including DNA ligase. Therefore, it is unlikely that the factor in S-3 h cytosol which restores DNA synthesis in drugtreated nuclei is DNA polymerase a or DNA ligase. The fact that nuclear extract from S-3 h nuclei restored DNA synthesis in drug-treated homogenate b u t not in drug-treated nuclei indicates that the factor exists also in nuclei and that it acts in co-operating with 'cytoplasmic factor(s)'. DNA synthesis in drug-treated nuclei induced by S-3 h cytosol has been shown to require the four deoxyribonucleoside triphosphates, Mg 2÷ and ATP, and to proceed discontinuously. The fact that the DNA synthesized in the system distributed in the half heavy density region indicates that the DNA synthesis is a continuation of the synthesis at the sites where it has been already in progress in vivo. These observations seem to suggest that the DNA synthesis in the complementation system is replication type. The recovery of DNA in a shoulder of the half heavy peak toward the light density region (Fig. 3B) offers t w o possibilities, that is, initiation of new replicon and repair synthesis. Repair synthesis is generally believed n o t to require ATP. In fact, our previous observations showed that ultraviolet-irradiated repair synthesis in HeLa cell homogenate did not require ATP [29]. Therefore, it is likely that the recovery of DNA in the light density region is due to the initiation of new replicon, even though the possibility that it is due to repair synthesis requiring ATP cannot still be excluded. Although the reason w h y there is a discrepancy between the results obtained with in vivo DNA synthesis after relief of the inhibition and those with in vitro DNA synthesis in the complementation system is not clear yet, it seems to be possible that the efficiency of in vitro reconstitution of initiation system is much lower than that of elongation system. In spite of the above limitation, our system is still promising for identification and characterization of protein factors involved in eukaryotic DNA replication.
330
Acknowledgements The authors thank Dr. Susumu Ikegami for a generous gift of aphidicolin. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. References 1 Friedman, D.L. and Mueller, G.C. (1968) Biochim. Biophys. Acta 1 6 1 , 4 5 5 - - 4 6 8 2 Lynch, W.E., Brown, R.F., Umeda, T., Langreth, S.G. and Lieberman, I. (1970) J. Biol. Chem. 245, 39 11--3916 3 Krokan, H., BjCrklid, E. and Prydz, H. (1975) Biochemistry 14, 4 2 2 7 - - 4 2 3 2 4 Franser, J.M.K. and Huberman, J.A. (1978) Biochim. Biophys. Acta 520, 271--284 5 Seki, S. and Mueller, G.C. (1976) Biochim. Biophys. Acta 4 3 5 , 2 3 6 - - 2 5 0 6 Franke, B. and Hunter, T. (1974) J. Mol. Biol. 83, 99--121 7 Tseng, B.Y. and Goulian, M. (1975) J. Mol. Biol. 9 9 , 3 1 7 - - 3 3 7 8 Franke, B. and Hunter, T. (1975) J. Virol. 15, 97--107 9 Otto, B. and Reiehard, P. (1975) J. Virol. 1 5 , 2 5 9 - - 2 6 7 10 DePamphilis, M.L. and Berg, P. (1975) J. Biol. Chem. 250, 4 3 4 8 - - 4 3 5 4 11 Brewer, E.N. (1979) Biochim. Biophys. Acta 564, 154--161 12 Seki, S., LeMahieu, M. and Mueller, G.C. (1975) Biochim. Biophys. Acta 3 7 8 , 3 3 3 - - 3 4 3 13 Berger, N.A. and J o h n s o n , E.S. (1976) Biochim. Biophys. Acta 425, 1--17 14 Hunter, T. and Franke, B. (1974) J. Mol. Biol. 8 3 , 1 2 3 - - 1 3 0 15 Tseng, B.Y. and Goulian, M. (1975) J. Mol. Biol. 9 9 , 3 3 9 - - 3 4 6 16 Kidwell, W.R. and Mueller, G.C. (1969) Biochem. Biophys. Res. C o m m u n . 3 6 , 7 5 6 - - 7 6 3 17 Krokan, H., Wist, E. and Prydz, H. (1977) Biochem. Biophys. Res. C ommun. 75, 414--419 18 MaalCe, O. and Hanawalt, P.C. (1961) J. Mol. Biol. 3 , 1 4 4 - - 1 5 5 19 Hanawalt, P.C., MaalCe, O., Cummings, D.T. and Schaechter, M. (1961) J. Mol. Biol. 3, 156--165 20 Lark, K.G. and Renger, M. (1969) J. Mol. Biol. 42, 221--235 21 Fujiwara, Y. (1972) Cancer Res. 32, 2089--2095 22 Hori, T. and Lark, K.G. (1973) J. Mol. Biol. 77, 391--404 23 Hand, R. and Tamm, I. (1972) Virology 4 7 , 3 3 1 - - 3 3 7 24 Weintraub, H. and Holtzer, H. (1972) J. Mol. Biol. 66, 13--35 25 Gautschi, J.R. and Kern, R.M. (1973) Exp. Cell Res. 80, 15--26 26 Gautschi, J.R. (1974) J. Mol. Biol. 84, 223--229 27 Stimac, E., Housman, D. and Huberman, J.A. (1977) J. Mol. Biol. 1 1 5 , 4 8 5 - - 5 1 1 28 Ono, Y., E n o m o t o , T., Hanaoka, F. and Yamada, M. (1978) Gann 6 9 , 2 0 7 - - 2 1 2 29 Kato, H., Hanaoka, F , Nishimura, T., Yamada, K. and Yamada, M. (1980) Biochim. Biophys. Acta 606, 47--56 30 Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H. and Mano, Y. (1978) Nature 275, 458-460 31 Reinhard, P., Maillart, P., Schluchter, M., Gautschi, J.R. and Schindler, P~. (1979) Biochim. Biophys. Aeta 564, 141--153 32 Kumar, K.V. and Friedman, D.L. (1972) Nature New Biol. 239, 74--76 33 Ono, Y., E n o m o t o , T. and Yamada, M. (1979) Gann 7 0 , 5 2 7 - - 5 3 2 34 Taylor, E.W. (1965) Exp. Cell Res. 40, 3 1 6 - - 3 3 2 35 Weiss, B.G. (1969) J. Cell Physiol. 73, 85--90
Biochimica et Biophysica Acta, 653 (1981) 316--330 Elsevier/North-Holland Biomedical Press
BBA 99853 A SYSTEM OF DNA R E P L I C A T I O N IN HeLa NUCLEI T R E A T E D WITH INHIBITORS OF P R O T E I N SYNTHESIS
KYOSUKE NAGATA, TAKEMI ENOMOTO and MASA-ATSU YAMADA Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113 (Japan) (Received January 19th, 1981 ) Key words: DNA synthesis; Cycloheximide; Puromycin; Cell cycle; (HeLa cell)
Summary An in vitro DNA synthesizing system consisting of isolated nuclei from HeLa cells which had been treated with inhibitors of protein synthesis was investigated. T r e a t m e n t with b o t h 30 pg/ml cycloheximide and 10 pg/ml p u r o m y c i n of S-phase cells reduced the rate of DNA synthesis immediately; however, the overall DNA synthesis c o n t i n u e d for up to 4 h with a diminished rate and then ceased. In the nuclei which were isolated from the cells which had been incubated with these drugs for 6 h, little i ncorporat i on of [3H]TTP into acidinsoluble materials was observed. Addition o f cytosol prepared from cells actively synthesizing DNA induced t h e in co r p o r atio n of [3H]TTP in these nuclei, while little induction was observed by the addition of cytosol prepared from drug-treated cells in spite of the fact th at the latter cytosol stimulated DNA synthesis in isolated nuclei f r o m n o n - tr eated cells. T he induced DNA synthesis was shown to require Mg 2+, all four deoxyribonucleoside triphosphates and ATP, and to proceed discontinuously. The activity inducing DNA synthesis in drug-treated nuclei fluctuated with the phases in a cell cycle and it was n o t ascribed solely to DNA polymerase a nor to DNA ligase.
Introduction DNA replication in mammalian cells is a com pl ex process t hat requires multiple approaches for the elucidation of its molecular mechanism. Up to the preAbbreviations: SDS, sodium dodecyl sulfate; SSC, standard saline/citrate solution (0.15 M NaC1/0.15M sodium citrate); AraCTP, cytosine-~-D-arabinofuranoside-5' triphosphate; BrdUrd, 5-bromo-2'-deoxyuridine; FdUrd, 5-fluoro-2'-deoxyuridine;dThd, thymidine;ddTTP,dideoxy 5'-triphosphate. 0005-2787/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
317 sent, two approaches have been used to study DNA synthesis in mammalian cells. One is in vitro study with purified enzymes. Such an approach should be important to ascertain the properties of individual components of DNA replication machinery, but it is not per se sufficient for understanding DNA replication occurring in intact cells. The other approach is to utilize a subcellular in vitro DNA-synthesizing system, involving cell lysates, isolated nuclei [1--11] and permeable cells [12,13]. These systems support intact replication machineries with endogenous template, enzymes and factors and are able to synthesis DNA in vitro with in vivo fidelity. They have been applied to the study on the mechanisms of DNA replication, such as discontinuous replication [6,7], R N A priming synthesis [14,15] and so on. In addition, these in vitro systems could be used for the identification and the characterization of protein factors participating in the processes of DNA replication because of their advantage of presenting no significant barrier to exogenously added macromolecules. However, little is known a b o u t the factors as yet except for so-called 'cytoplasmic factor(s)', which stimulate DNA synthesis in isolated nuclei by accelerating the formation and ligation of primary short fragments {Okazaki pieces) [4,7--9,16,17]. It has been shown from experiments with various organisms that there exists a close coupling between DNA replication and protein synthesis. Studies with bacteria have shown that protein synthesis is a prerequisite for the initiation of a new cycle of DNA replication b u t is not required for the continuation [18-20]. In mammalian cells, it is observed that inhibition of protein synthesis causes a rapid decrease in DNA synthesis before a replication cycle is completed. The rapid decrease is suggested to be due to the inhibition of the initiation of new replicon [21--23] and the decrease in the DNA chain elongation rate at the replicon already in progress [24--27]. Therefore, it seems likely that newly synthesized proteins, which are turned over rapidly, are required for the above processes. Some of the major advances in understanding the roles of factors in DNA replication of prokaryotic cells came from the works using complementation tests of extracts from m u t a n t cells deficient in the factors to be examined. Unlike prokaryotes, DNA replication mutants of eukaryotic cells are n o t available yet, with the exception of yeast cell division cycle mutants. In order to overcome the above limitiation, we have a t t e m p t e d to establish an in vitro system corresponding to mutants. In this communication we will report complementation of the nuclei which are isolated from the cells treated in vivo with inhibitors of protein synthesis and are deficient in some protein factors required for DNA synthesis, with cytosol which is prepared from cells actively synthesizing DNA. It has been suggested that the cytosol prepared from cells actively synthesizing DNA contains the factor{s) which is different from 'cytoplasmic factor', being detectable only b y use of the nuclei isolated from the cells treated in vivo with inhibitors of protein synthesis. Materials and Methods Cell culture and synchronization. HeLa $3 cells were maintained in monolayer culture in Eagle's minimum essential medium supplemented with 10% calf
318 serum (growth medium) at 37°C. Synchronization was accomplished by the addition of 1 mM h y d r o x y u r e a for 16 h to accumulate the cells at the G1/S boundary. Cells were washed twice with Mgz÷, Ca2÷-free phosphate-buffered saline to release the block and were incubated in the fresh growth medium. Treatment with inhibitors of protein synthesis. At 30 min incubation after the release from the h y d r o x y u r e a block, puromycin and cycloheximide were added at a final concentration of 10 and 30 t~g/ml, respectively, and cells were further incubated for 6 h. Cells were harvested as described in the following section except that trypsinization was carried out in the presence of inhibitors of protein synthesis. Preparation of cell lysate, nuclei, cytoplasm and cytosol. Cells were washed once with Mg 2÷, Ca2÷-free phosphate-buffered saline and 0.05% trypsin containing 0.02% EDTA, successively, and trypsinized with the same solution. The trypsinization was stopped by the addition of cold growth medium and cells were collected by low-speed centrifugation. Collected cells were washed twice with Mg z÷, Ca2÷-free phosphate-buffered saline and once with hypotonic medium containing 10 mM Tris-HC1, pH 7.5 and 2 mM MgCl~ and were stored at --20 or --80°C until use. After thawing cells, cell lysate, nuclei, cytoplasm and cytosol were prepared as described in previous papers [28,29]. Nuclei and cytosol prepared from cells treated with inhibitors of protein synthesis as described above were designated 'drug-treated nuclei' and 'drug-treated cytosol', respectively, and those prepared from cells harvested at 3 h after release from h y d r o x y u r e a block were designated 'S-3 h nuclei' and 'S-3 h cytosol', respectively. Preparation of nuclear salt extract. Nuclei were isolated as described with the exception that the media used contained no NaC1. Isolated nuclei were resuspended in buffer A (60 mM Tris-HC1, pH 8.5/11 mM glucose/0.05% Triton X-100/0.5 mM EDTA/1 mM 2-mercaptoethanol/1 mM MgC12) containing various concentrations of NaC1. After keeping for 60 min at 0°C, the nuclear suspension was centrifuged for 10 min at 2500 rev./min. The supernatant was designated 'nuclear salt extract'. The nuclear salt extract was used after dialysis against buffer A containing 80 mM NaC1. Labeling o f DNA in vivo. Cells were incubated with growth medium containing 0.5 pCi/ml [3H]dThd (70 Ci/mmol) for the indicated time. In the experiments requiring highly labeled DNA, cells were labeled with 10 pCi/ml [3H]dThd in Eagle's minimum essential medium supplemented with 10% dialyzed calf serum in the presence of both 1 • 10 -6 M amethopterin and 5 • 10 -s M adenosine or 5 • 10 -6 M FdUrd to decrease the endogenous thymidine pool. Assay of DNA synthesis in vivo. NaN3 was added at a final concentration of 50 pg/ml to the cells which were labeled with [3H]dThd. Cells were harvested as described above and suspended in Mg 2÷, Ca2+-free phosphate-buffered saline at a concentration of (6--10) • l 0 s cells/ml. 0.5 ml of 0.2% SDS was added to 0.5 ml of the cell suspension to lyse cells. An equal volume of 10% trichloroacetic acid/0.1 M sodium pyrophosphate was added to the lysate. The radioactivity incorporated into 5% trichloroacetic acid-precipitable materials was collected on Whatman GF/C glass-fiber filter, washed with 5% trichloroacetic acid five times and with ethanol and acetone once, successively. After drying the filter, the radioactivity was counted in a toluene-based scintillation solution
319 with a liquid scintillation spectrometer.
Assay o f DNA synthesis in in vitro nuclear system and DNA polymerase activity. The standard assay mixture with a total volume of 0.15 ml contained 50 mM Tris-HC1, pH 8.5, 4 0 m M NaC1, 5 mM MgC12, 5 mM 2-mercaptoethanol, 0.25 mM EDTA, 0.025% Triton X-100, 5.5 mM glucose, 3.3 mM ATP, 33 pM each dATP, dCTP, dGTP, 3.3 pM [3H]TTP (1 Ci/mmol) and 5 • 10 s nuclei with or without cytosol. The reaction was stopped by the addition of 0.85 ml of cold water and 1 ml of 10% trichloroacetic acid. The radioactivity was measured as above. DNA polymerase activity was measured as described previously [28].
Alkaline sucrose gradient centrifugation Cells. Cells were lysed with 4/3 vol. of SDS solution containing 0.5% SDS, 0.05 M Tris-HC1, pH 8.0, 0.15 M NaC1 and 0.015 M EDTA, and incubated at 37°C for 8 h with 1/4 vol. of pronase solution containing 10 mg/ml pronase E, 0.05 M Tris-HC1, pH 8.0, 0.15 M NaC1 and 0.015 M EDTA, which had been preheated at 80°C for 10 min and preincubated at 37°C for 2 h to inactivate DNAase. After the addition of NaOH to 0.1 M, samples were kept for 2 h at room temperature, and then layered on the top of 4.8 ml of a 5--20% sucrose gradient containing 0.1 M NaOH, 0.9 M NaC1 and 0.01 M EDTA and centrifuged for 2.5 h at 106 600 X g in a Hitachi RPS-40T2 rotor at 16°C. The fractions were collected from the b o t t o m of the tube. To each fraction, 200 #g of herring sperm DNA and an equal volume of 10% trichloroacetic acid were added. Acid-insoluble radioactivity was collected on the glass-fiber filter. Nuclei. 1 • 104 nuclei were incubated in 0.3 ml of standard reaction mixture except that the specific activity of [3H]TTP was 3 Ci/mmol. After the incubation for various times at 37~C, samples were treated as described above and loaded on 29 ml of 5--20% sucrose gradients containing 0.1 M NaOH 0.9 M NaC1 and 0.01 M EDTA. Centrifugation was carried out in a Hitachi RPS25 rotor at 51 500 X g for 12 h at 16°C.
Neutral CsCl equilibrium density gradient centrifugation Cells. Cells were lysed in 1 ml of 50 ~g/ml proteinase K solution containing 10 mM Tris-HC1, pH 8.0, 10 mM NaC1, 10 mM EDTA and 0.5% SDS, and incubated for 12 h at 37°C. The lysate was dialyzed for 8 h against 0.01 X SSC, pH 7.0. The dialyzate was adjusted to 10 mM Tris-HC1, pH 8.0, to be digested with 1 mg RNAase A at 37°C for 30 min. After sonication with a Bronson sonifier (at 50 W, 5-times 15-s), 0.3 ml of the aliquot was diluted to 3 ml with 0.01 M Tris-HCl, pH 8.0. 4 g of solid CsC1 was dissolved. The sheared DNA was centrifuged for 65 h at 129 000 X g in a Hitachi RPS 40T2 rotor at 20°C. The fractions were collected from the b o t t o m of the tube. Nuclei. 2 • 106 nuclei were incubated in 0.6 ml of standard reaction mixture except that the specific activity of [3H]TTP was 3 Ci/mmol. To the sample, 0.2 ml of buffer K, containing 40 mM Tris-HC1, pH 8.0, 40 mM NaC1 and 2% SDS, and 0.2 ml of 0.25 mg/ml proteinase K, in 10 mM Tris-HC1, pH 8.0, 10 mM NaC1, 10 mM EDTA and 0.5% SDS, were added successively and processed as described above.
320
Materials. [3H]TTP (47--59 Ci/mmol), [3H]dThd (70 Ci/mmol) and [14C]dThd (57 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, U.K.; ATP, dATP, dCTP, dGTP, TTP and proteinase K were from Boehringer Mannheim; adenosine, dThd, amethopterin, h y d r o x y u r e a and AraCTP from Sigma; CsC1 from Merck; RNAase A from Worthington; pronase E from Kaken-Kagaku; Japan; cycloheximide from Wako-Junyaku, Japan; puromycin from Makor Chemical. Results
Inhibition o f DNA synthesis by inhibitors of protein synthesis To examine the effect of inhibitors of protein synthesis on DNA synthesis in vivo, cells were incubated in the presence of varying concentrations of puromycin or cycloheximide for 1 h and then the incorporation of [3H]dThd for 5 min was measured (Fig. 1A and B). The incorporation was inhibited by 90% at a concentration of 30 pg/ml puromycin or 3 pg/ml cycloheximide. Although cycloheximide inhibited more effectively than puromycin, the degree of inhibition increased scarcely with concentrations of cycloheximide higher than 3 pg/ml. Fig. 2 illustrates the rate of DNA synthesis in the cells which were incubated for various periods in the presence of 10 pg/ml cycloheximide and 30 pg/ml puromycin. The rate fell immediately, and within 5 min after the addition of drugs incorporation was reduced by 70%. Thereafter, the degree of inhibition increased gradually up to 1 h and, upon prolonged incubation, a fairly constant rate of incorporation, about 5% of control, was observed. The other combination of 30 pg/ml cycloheximide and 10 pg/ml puromycin gave a similar result. We used hereafter this combination of drugs to inhibit DNA synthesis. Fig. 3 shows the cumulative curve of DNA synthesis in the presence or absence of both drugs. The incorporation of thymidine in drug-treated cells continued at a diminished rate for up to 4 h and no further incorporation was observed after treatment for 6 h. Analysis o f DNA synthesis in the presence o f inhibitors Evidence from previous reports suggests that the inhibition of DNA synthesis by cycloheximide does not result from the alteration of precursor metabolism [24,25,27]. The reduction of DNA synthesis in drug-treated cells, therefore, may be speculated to be due to the reduction of the rate of chain elongation [24--26] and/or the inhibition of initiation of new replicon [21,22]. To obtain more information a b o u t our system, synthesized DNA was analyzed. Cells were pulse-labeled for 2 min with [3H]dThd at the beginning of S phase and chased in the presence of inhibitors. As shown in Fig. 4, it t o o k 3 h for the short chain to elongate to bulk DNA in the presence of inhibitors, while, in control cells, elongation to bulk DNA was accomplished within 0.5--1 h (not shown). Little decrease of total count was observed during the chase in the presence of inhibitors. To examine the effect of inhibitors on the initiation on new replicon, cells were incubated with BrdUrd for 6 h in the presence of inhibitors and then washed to be freed from inhibitors, and then pulse-labeled with [3H]dThd. Synthesized DNA was analyzed by neutral CsC1 density gradient centrifugation.
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Fig. 1. Effect o f inhibitors o f p r o t e i n s y n t h e s i s o n D N A synthesis. 1 • 10 5 cells w e r e i n c u b a t e d for 2 h after release f r o m the h y d r o x y u r e a b l o c k , and further i n c u b a t e d for 1 h in the presence o f v a t t i n g c o n c e n t r a t i o n o f p u r o m y c i n ( A ) or c y c l o h e x i m i d e (B) and then pulse-labeled for 5 rain w i t h 0.5 /~Ci/ml [3H]dThd: Fig. 2. Kinetics of the i n h i b i t i o n o f D N A s y n t h e s i s b y c o m b i n a t i o n o f c y c l o h e x i m i d e and p u r o m y c i n . Cells w e r e i n c u b a t e d for 2 h after release f r o m the h y d r o x y t t r e a b l o c k and further i n c u b a t e d for i n d i c a t e d p e r i o d in the presence o f b o t h 10 p g / m l c y c l o h e x i m i d e and 3 0 p g / m l p u r o m y c i n and t h e n pulse-labeled for 5 rain w i t h [ 3 H ] d T h d . Fig. 3. T i m e course o f c u m u l a t i v e i n c o r p o r a t i o n of [ 3 H ] d T h d . Cells w e r e i n c u b a t e d for 30 rain after release f r o m the h y d r o x y u r e a b l o c k and t h e n t h e y w e r e labeled w i t h 0.5 p C i / m l [ 3 H ] d T h d in the absence ( o ) o r presence ( e ) o f b o t h 3 0 pg/rnl c y c l o h e x i m i d e and 10 p g / m l p u r o m y c i n for time i n d i c a t e d in the figure.
Most of D N A synthesized after 10 min incubation banded at the light density region (Fig. 5A) and a shift to the half heavy density region was observed after 30 min incubation {Fig. 5B). DNA synthesis in vitro in isolated nuclei treated with inhibitors in vivo or in vitro
To examine whether the inhibition of D N A synthesis by the protein synthesis inhibitors resulted from a direct interaction of the drugs with components of the replication machinery or from the inhibition of protein synthesis, D N A synthesis in isolated nuclei treated with the inhibitors in vitro or in vivo was measured. The D N A synthesis in the nuclei isolated from cells actively synthesizing D N A was not inhibited by in vitro treatment of both 30 #g/ml cycloheximide and 10 ~g/ml puromycin, while a reduction in DNA-synthesizing
322
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Fig. 4. E f f e c t o f i n h i b i t o r s o f p r o t e i n s y n t h e s i s o n D N A c h a i n e l o n g a t i o n . S y n c h r o n i z e d cells w e r e f r e e d f r o m t h e h y d r o x y u r e a b l o c k a n d i m m e d i a t e l y p u l s e - l a b e l e d f o r 2 m i n (~) in t h e a b s e n c e o f inhibitors. A f t e r pulse-labeling, t h e cells w e r e w a s h e d w i t h Mg 2+, Ca2+-free p h o s p h a t e - b u f f e r e d saline a n d c h a s e d w i t h 1 • 10 -S M u n l a b e l e d d T h d for 1 h (~) a n d 3 h ( e ) in t h e p r e s e n c e o f b o t h 3 0 pg/ml c y c l o h e x i m i d e a n d 1 0 pg/ml p u r o m y e i n . D N A s w e r e p r e p a r e d as d e s c r i b e d in Materials a n d M e t h o d s a n d s u b j e c t e d to 5 - - 2 0 % alkaline s u c r o s e g r a d i e n t centrifugation. Sedimentation w a s f r o m r i g h t to l e f t . T h e a r r o w d e n o t e s t h e c o r r e s p o n d i n g p o s i t i o n o f b u l k D N A p r e p a r e d f r o m cells u n i f o r m l y l a b e l e d f o r 24 h. Fig. 5. N e u t r a l CsCI g r a d i e n t analysis o f D N A s y n t h e s i z e d a f t e r release f r o m i n h i b i t i o n of p r o t e i n synthesis. T h e cells p r e - l a b e l e d w i t h [ 14 C ] d T h d w e r e s y n c h r o n i z e d w i t h h y d r o x y u r e a . A f t e r release f r o m t h e b l o c k , cells w e r e i n c u b a t e d in fresh g r o w t h m e d i u m c o n t a i n i n g 5 • 10 -$ M B r d U r d a n d 5 • 10 -6 M F d U r d ( h e a v y m e d i u m ) . 30 rain a f t e r t h e i n c u b a t i o n , c y c l o h e x i m i d e a n d p u r o m y c i n w e r e a d d e d a t c o n c e n t r a t i o n s of 30 a n d 10 p g / m l , r e s p e c t i v e l y . I n c u b a t i o n w a s c o n t i n u e d for a n o t h e r 5.5 h in t h e h e a v y m e d i u m . Cells w e r e w a s h e d w i t h Mg 2+, Ca2+-free p h o s p h a t e - b u f f e r e d saline a n d p u l s e - l a b e l e d w i t h 2 p C i / m l [ 3 H ] d T h d f o r 1 0 r a i n ( A ) o r 3 0 rain (B) in fresh g r o w t h m e d i u m . • 14C p r e - l a b e l e d ; c , 3 H pulse-labeled.
activity in isolated nuclei was noted when nuclei were isolated from the cells treated with inhibitors in vivo (Fig. 6). The in vitro D N A synthesizing activity was not changed till a 30 min treatment of drugs in vivo. Thereafter, it decreased gradually for up to 4 h, and little activity was observed after 6 h treatment.
Complementation of nuclei with cytosol Nuclei deficient in some proteins involved in D N A replication may become an useful tool for identification and evaluation of such proteins. We next performed complementation experiments. Drug-treated nuclei or S-3 h nuclei were complemented with a cytosol prepared from cells actively synthesizing D N A (S-3 h cells). Fig. 7 shows dose response curve of S-3 h cytosol. [3H]TTP incorporation in both nuclei were enhanced by S-3 h cytosol. In S-3 h nuclei, the incorporation was increased with the amount of cytosol up to three cell equivalents and reached a plateau at higher concentrations. In the case of drugtreated nuclei, the incorporation increased proportionally to the amount of S-3 h cytosol up to five cell equivalents. Fig. 8 shows kinetics of the incorporation of [3H]TTP in drug-treated nuclei complemented with three or five cell equivalents of cytosol. The incorporation in complemented nuclei increased
323
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Fig. 6. I n v i t r o D N A - $ y n t h e s i z i n g a c t i v i t y in n u c l e i i s o l a t e d f r o m cells t r e a t e d w i t h i n h i b i t o r s o f p r o t e i n s y n t h e s i s . Cells s y n c h r o n i z e d w i t h h y d r o x y u r e a w e r e r e l e a s e d f r o m t h e b l o c k a n d i n c u b a t e d f o r 30 r a i n in fresh g r o w t h m e d i u m a n d t h e n p u r o m y c i n a n d c y c l o h e x i m i d e w e r e a d d e d a t c o n c e n t r a t i o n s o f 10 a n d 3 0 # g / m l , r e s p e c t i v e l y . Cells w e r e i n c u b a t e d in t h e p r e s e n c e o f d r u g s for t h e p e r i o d s i n d i c a t e d a n d n u c l e i w e r e i s o l a t e d f r o m t h e cells. I s o l a t e d n u c l e i (1 • 1 0 6 ) w e r e i n c u b a t e d in t h e s t a n d a r d r e a c t i o n m i x t u r e f o r 2 0 rain at 37~C. Fig. 7. E f f e c t o f S-3 h c y t o s o l o n D N A s y n t h e s i s in S-3 h a n d d r u g - t r e a t e d nuclei. S-3 h a n d d r u g - t r e a t e d n u c l e i w e r e i s o l a t e d f r o m S-3 h a n d d r u g - t r e a t e d cells, r e s p e c t i v e l y , as d e s c r i b e d in Materials a n d M e t h o d s . S-3 h c y t o s o l w a s p r e p a r e d f r o m S-3 h c y t o p l a s m as t h e s u p e r n a t a n t b y c e n t r i f u g a t i o n a t 1 0 5 0 0 0 × g f o r 60 rain. 5 • 105 n u c l e i (o, S-3 h nuclei; o, d r u g - t r e a t e d n u c l e i ) w e r e i n c u b a t e d w i t h S-3 h c y t o s o l in a n a m o u n t f r o m 1 t o 5 cell e q u i v a l e n t s in the s t a n d a r d r e a c t i o n m i x t u r e f o r 20 rain a t 3 7 ° C . E a c h v a l u e w a s n o r m a l i z e d as t h e i n c o r p o r a t i o n p e r 1 • 106 nuclei.
with incubation time up to 5--10 min and slightly upon a continued incubation. Little incorporation was observed in non-complemented nuclei. Table I shows complementation of nuclei with drug-treated cytosol as well as that with S-3 h cytosol. It must be noted that drug-treated cytosol, which had no effect on drug-treated nuclei, increased the incorporation in S-3 h nuclei to nearly the same level as S-3 h cytosol.
Characterization o f DNA synthesis in drug-treated nuclei induced by S-3 h cy tosol To characterize DNA synthesis induced in drug-treated nuclei, the requireTABLE I D N A S Y N T H E S I S IN N U C L E I C O M P L E M E N T E D W I T H C Y T O S O L 5 " 105 n u c l e i w e r e i n c u b a t e d f o r 10 rain at 3 7 ° C in t h e a b s e n c e or p r e s e n c e o f d r u g - t r e a t e d c y t o s o l o r S - 3 h c y t o s o l o f 5 cell e q u i v a l e n t s ( 2 . 5 • 106 cells). E a c h v a l u e in t h e t a b l e i n d i c a t e s t h e i n c o r p o r a t i o n / 1 • 106 nuclei. Nuclei
Cytosol
T M P incorporated (pmol/1 • 10 6 nuclei)
Drug-treated Drug-treated Drug-treated S-3 h S-3 h S-3 h
none drug-treated S-3 h none drug-treated S-3 h
0.62 0.32 2.10 1.02 3.33 3.66
324 m e n t of the system and the effect of several inhibitors were investigated. As shown in Table II, the system required all four deoxyribonucleoside triphosphates, Mg 2÷ and ATP. The optimal level of ATP under the conditions tested was around 3--5 mM. Aphidicolin, which is a specific inhibitor for DNA polymerase ~ [30], inhibited DNA synthesis in the complementation system by 75% at 6/~g/ml. DNA synthesis was also sensitive to AraCTP b u t n o t sensitive to ddTTP. Fig. 9 shows alkaline sucrose gradient centrifugation analysis of DNA synthesized in the complementation system. A b o u t 50% of the radioactivity was recovered in short fragments by 1 min pulse-labeling and the rest was distributed widely in longer DNA chains. When pulse-labeled for 5 min, a marked increase of the radioactivity incorporated into longer DNA chains was observed. After 20 min pulse-labeling, almost all the radioactivity appeared in the longer DNA. The short fragments in the peak near the top of the gradient and longer DNA in the broad peak appearing in the two-thirds from the b o t t o m of the gradient were calculated as being about 200 nucleotide long DNA and 40 S DNA, respectively, using nucleosome m o n o m e r DNA and X phase DNA as markers. To check that DNA synthesized initially as short fragments is a precursor to be elongated to longer DNA, pulse-chase experiments were carried o u t (Fig. 10). By 12.5 min chasing, the radioactivity in the peak of short fragments decreased and migrated to the somewhat longer fraction and the majority of the radioactivity distributed in 40 S DNA. After 17.5 min chase, the radioactivity in both peaks of short fragments decreased and almost all the radioactivity was found in 40 S DNA. In order to characterize DNA synthesis in the complementation system further, synthesized DNA was analyzed by neutral CsC1 equilibrium density gradient centrifugation. Nuclei were isolated from cells which were incubated with BrdUrd during the treatment of the inhibitors of protein synthesis and were
TABLE II REQUIREMENTS OF DNA SYNTHESIS IN DRUG-TREATED NUCLEI COMPLEMENTED WITH S-3 h CYTOSOL
T h e c o m p l e t e s y s t e m w a s as d e s c r i b e d in Materials and M e t h o d s . 5 at 3 7 ° C for 2 0 m i n w i t h S - 3 h c y t o s o l o f 4 c e l l e q u i v a l e n t s . o m i s s i o n o f A T P a n d M g 2 + , S - 3 h c y t o s o l w a s u s e d at a m o u n t o f 5 assay m i x t u r e and i n h i b i t o r s w e r e o m i t t e d and a d d e d , r e s p e c t i v e l y , a s bated
Condition
Percent o f complete system
Complete system
100
-- ATP - - M g 2+
4 0
-- dATP -- dGTP
27 18
-- dCTP
14
-- dATP, -- dGTP -- dATP,-dCTP
16 16
-- dCTP,-dGTP + Aphidicolln (0.2/~g/ml) + Aphidicolin (6 / ~ g / m l )
10 86 25
+ AraCTP
19
(1 raM)
• 105 drug-treated nuclei were incuIn t h e case o f the e x p e r i m e n t s w i t h cell equivalents. C o m p o n e n t s o f the g i v e n in the t a b l e .
325 lo I
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Fig. 8. T i m e c o u r s e o f D N A s y n t h e s i s in d r u g - t r e a t e d n u c l e i c o m p l e m e n t e d w i t h S-3 h c y t o s o l . Drugt r e a t e d n u c l e i (5 • 1 0 s ) w e r e i n c u b a t e d at 3 7 ° C for i n d i c a t e d p e r i o d s in the standard r e a c t i o n m i x t u r e in t h e a b s e n c e ( o ) or p r e s e n c e o f S-3 h c y t o s o l o f 3 cell e q u i v a l e n t s (A) and 5 cell e q u i v a l e n t s ( e ) . Fig. 9. A l k a l i n e s u c r o s e s e d i m e n t a t i o n analysis o f D N A s y n t h e s i z e d in in vitro c o m p l e m e n t a t i o n s y s t e m . Cells s y n c h r o n i z e d w i t h h y d r o x y u r e a w e r e r e l e a s e d fTom the b l o c k and i n c u b a t e d for 3 0 rain in fresh g r o w t h m e d i u m and t h e n c y c l o h e x i m i d e and p u r o m y c i n w e r e a d d e d at c o n c e n t r a t i o n s o f 3 0 and 1 0 # g / m l , r e s p e c t i v e l y . N u c l e i w e r e i s o l a t e d f r o m t h e cells ( d r u g - t r e a t e d n u c l e i ) . S-3 h c y t o s o l w a s prepared f r o m cells which were synchronized a n d released into S phase for 3 h. I • I06 drug-treated nuclei were incubated with S-3 h cytosol 5 cell equivalents (5 • 106) for 1 m i n (©), 5 rain (e) and 20 m i n (A) at 3 7 ° C in the standard reaction mixture except that the specific activity of [ 3 H ] T T P w a s 3 Ci/mmol. Sedimentation was from right to left.
labeled with [3H]TTP in vitro. DNA synthesized in non-complemented nuclei banded in the half heavy density region, suggesting that DNA synthesis in the drug-treated nuclei is the continuation of DNA replication at replicons which have been already in progress in vivo (Fig. l l A ) . In the complementation system, synthesized DNA distributed in the peak of half heavy density with a shoulder toward the light density region (Fig. l l B ) . Subcellular distribution of the activity S-3 h cytoplasm was fractionated by centrifugation into several fractions. The complementing activity to drug-treated nuclei was observed both in the cytoplasm and the postmitochondrial fraction (Table III, lines 2 and 4). However, the former fraction incorporated [3H]TTP considerably by itself. This incorporation may be derived from mitochondrial DNA synthesis (Table III, line 3). The activity in the postmitochondrial fraction was further divided into cytosol and microsomal fraction (Table III, lines 5 and 6). Cytosol alone had no capacity to incorporate [3H]TTP into acid-insoluble materials and almost all the complementing activity was recovered in this fraction. Fluctuation of the activity during a cell cycle It has been reported that DNA synthesis in isolated nuclei is stimulated by
326
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Fig. i0. Alkaline sucrose sedimentation analysis of D N A pulse-labeled and chased in in vitro c o m p l e m e n tation system. Drug-treated nuclei and S-3 h cytosol were prepared as described in the legend to Fig. 9. 1 • 106 drug-treated nuclei were pulse-labeled in the presence of S-3 h cytosol of 5 cell equivalents for 2.5 m i n (o) and chased with a 100-fold excess of unlabeled T T P for 12.5 rain (A) or 17.5 rain (o). Sedimentation was from right to left. Fig. 11. N e u t r a l CsC1 e q u i l i b r i u m d e n s i t y g r a d i e n t analysis of D N A s y n t h e s i z e d in in v i t r o c o m p l e m e n t a t i o n s y s t e m . N u c l e i w e r e i s o l a t e d f r o m t h e cells t r e a t e d as d e s c r i b e d in t h e l e g e n d t o Fig. 5 . 2 • 106 d r u g t r e a t e d n u c l e i w e r e i n c u b a t e d in t h e s t a n d a r d r e a c t i o n m i x t u r e , e x c e p t t h a t t h e specific a c t i v i t y o f [ 3 H ] T T P w a s 3 C i / r a r a o l , f o r 10 rain at 3 7 ° C w i t h o u t ( A ) o r w i t h (B) S-3 h c y t o s o l of 5 cell e q u i v a l e n t s , o, 14C pre-labeled~ o, [ 3 H ] T T P - l a b e l e d .
cytoplasm (cytoplasmic factor), regardless of the phase of cells from which the cytoplasm is prepared [ 1 , 1 1 , 3 1 , 3 2 ] . We have also confirmed this by using S-3 h nuclei and cytoplasms prepared from cells in various phases of a cell cycle. To ascertain whether or not phase-dependency is observed in the case of the
TABLE III S U B C E L L U L A R D I S T R I B U T I O N OF C O M P L E M E N T I N G A C T I V I T Y Cytoplasm, mitochondrial fraction, postraitochondrial fraction, cytosol and raicrosomal fraction were p r e p a r e d f r o m S-3 h cells. M i t o c h o n d r i a l a n d p o s t m i t o c h o n d r i a l f r a c t i o n s w e r e p r e p a r e d f r o m c y t o p l a s m b y c e n t r i f u g a t i o n f o r 2 0 rain at 12 0 0 0 × g. C y t o s o l a n d r a i c r o s o m a l f r a c t i o n w e r e p r e p a r e d f r o m p o s t r a i t o c h o n d r i a l f r a c t i o n b y c e n t r i f u g a t i o n f o r 6 0 rain a t 1 0 5 0 0 0 × g. M i t o c h o n d r i a l a n d r a i c r o s o r a a l fract i o n s w e r e s u s p e n d e d in n u c l e a r i s o l a t i o n b u f f e r . 5 • 105 n u c l e i w e r e i n c u b a t e d f o r 2 0 rain in t h e p r e s e n c e o f 5 cell e q u i v a l e n t s o f e a c h f r a c t i o n . T h e v a l u e in t h e p r e s e n c e o f n u c l e i i n d i c a t e s t h e i n c o r p o r a t i o n p e r 1 • 105 n u c l e i . TMP i n c o r p o r a t e d ( p m o l )
1 2 3 4 5 6
None Cytoplasm Mitochondrial fraction Postraitochondrial fraction Cytosol Microsomal fraction
Fraction only
With d r u g - t r e a t e d n u c l e i
-5.15 4.00 1.49 0.00 0.74
1.27 17.49 6.78 10.42 7.45 2.85
327
complementing activity, cells were synchronized and cytosols were prepared at various phases of a cell cycle. Fig. 12 shows the fluctuation pattern of the activity. The S phase began immediately following release from the hydroxyurea block and lasted for 8--9 h. From 9 to 12 h, the majority of the cells entered into G2 phase and at 15 h the population had almost doubled. The activity was highest in early S phase and decreased gradually with the progression of cell cycle from S to G2 phase. Does the complementing activity arise from a known enzyme? As reported in our previous papers [28,33], most of the DNA polymerase activity in cytosol is derived from DNA polymerase a. After treatment of the inhibitors of protein synthesis for 6 h, the DNA polymerase activity in the cytosol reduced to 60% of the original level. If the complementing activity arises from DNA polymerase a, then a large amount of drug-treated cytosol should restore DNA synthesis in drug-treated nuclei. This was not the case. As indicated in Table I, even five cell equivalents of drug-treated cytosol had no effect on the DNA synthesis in drug-treated nuclei. Furthermore, highly purified DNA polymerase a did not affect the DNA synthesis (not shown). To ascertain that the complementing activity arises from enzymes necessary for the synthesis of high molecular weight DNA, including DNA ligase, we next examined whether or not drug-treated cytosol supports the synthesis of high molecular weight DNA. S-3 h nuclei were incubated with drug-treated cytosol and the product was analyzed by alkaline sucrose gradient centrifugation. In the absence of drug-treated cytosol, little high molecular weight DNA was syn"i 2.5 2C i
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Fig. 12. F l u c t u a t i o n o f c o m p l e m e n t i n g a c t i v i t y during a cell c y c l e . CeLls w e r e s y n c h r o n i z e d w i t h h y d r o x y urea at G 1 / S b o u n d a r y . C y t o s o l s w e r e p r e p a r e d b y h a r v e s t i n g t h e cells at t h e 3-h intervals a f t e r release f r o m t h e b l o c k . T h e a c t i v i t y o f e a c h e y t o s o l w a s a s s a y e d in t h e standard r e a c t i o n m i x t u r e at 37 ° C f o r 2 0 rain b y using d r u g - t r e a t e d n u c l e i . D r u g - t r e a t e d n u c l e i i n c o r p o r a t e d 0 . 4 4 p m o l o f [ 3 H ] T M P b y t h e m s e l v e s . Fig. 1 3 . A l k a l i n e s u c r o s e s e d i m e n t a t i o n analysis o f D N A s y n t h e s i z e d in S-3 h n u c l e i c o m p l e m e n t e d w i t h d r u g - t r e a t e d c y t o s o l . S-3 h n u c l e i and d r u g - t r e a t e d c y t o s o l w e r e p r e p a r e d as d e s c r i b e d in M a t e r i a l s and M e t h o d s . 1 • 1 0 6 S-3 h n u c l e i c o m p l e m e n t e d w i t h d r u g - t r e a t e d c y t o s o l o f 3 cell e q u i v a l e n t s w e r e incub a t e d in t h e standard r e a c t i o n m i x t u r e e x c e p t t h a t t h e s p e c i f i c a c t i v i t y o f [ 3 H ] T T P w a s 3 C i / m m o l , for 1 rain ( o ) and c h a s e d w i t h a 1 0 0 - f o l d e x c e s s o f u n l a b e l e d TTP for 1 0 rain ( o ) . S e d i m e n t a t i o n w a s f r o m right to left.
328 thesized (not shown), while in its presence a marked increase in the synthesis of high molecular weight DNA was observed, and the short fragments synthesized during 1 min pulse-labeling were chased to high molecular weight DNA during another 10 min incubation (Fig. 13).
Does the complementing factor exist in nuclei? S-3 h nuclei were prepared using media containing no NaC1, and nuclear salt extract was prepared as described under Materials and Methods. Addition of the extract to drug-treated nuclei caused no effect on the DNA synthesis, whereas, in the drug-treated homogenate system consisting of drug-treated nuclei and drug-treated cytoplasm, DNA synthesis was restored by the addition of the nuclear salt extract. The activity was extracted efficiently with 80 mM as well as 250 mM NaC1 in the presence of 60 mM Tris-HC1. The combination of 80 mM NaC1 and 60 mM Tris-HC1 is the same condition as that used for the usual preparation of nuclei. Discussion The results presented here show that DNA synthesis in mammalian cells requires concomitant protein synthesis, in agreement with reports by others [21--27,34,35]. The difference between the time course of inhibition of DNA synthesis in vivo (Fig. 2) and in vitro (Fig. 6) may be due to the difference between the systems in that DNA synthesis in isolated nuclei is performed by proteins and enzymes remaining in the nuclei; therefore the situation for DNA synthesis in isolated nuclei, even in non-drug-treated nuclei, is closer to that of drug-treated cells rather than non-drug-treated cells. The behavior of DNA synthesized in the cells freed from inhibitors on neutral CsC1 gradient analysis (Fig. 5) can be explain by the assumption that DNA synthesis restored in the cells occurs predominantly in new replicons and that it takes about 30 min for new DNA chains to elongate in order to be linked with heavy DNA chains which have been synthesized in the presence of inhibitors, or that initiation of new replicons can occur immediately after removal of inhibitors but restoration of DNA synthesis at pre-existing replication forks takes time. Therefore, it seems likely that the combination of cycloheximide and puromycin causes the reduction of both the rate of chain elongation (Fig. 4) and frequency of initiation of new replicon. From the experiments using isolated nuclei, it is unlikely that the reduction of DNA synthesis by the inhibitors is due to the change of metabolism of precursors or to a direct effect of the drugs on replication machinery. Rather, the rapid reduction of DNA synthesis following the addition of the inhibitors is due to the depletion of enzymes a n d / o r proteins which are synthesized in conjunction with DNA synthesis and is turned over rapidly. Therefore, isolated nuclei prepared from the cells treated with the inhibitors may be deficient in some proteins necessary for DNA replication and can be considered as a sort of m u t a n t . By use of such nuclei, it became possible to search for protein(s) required stringently for DNA replication and turned over rapidly, in the cytosol from the cells activity synthesizing DNA. It has been reported that cytoplasm of mammalian cells contains 'cytoplas-
329 mic factor' which stimulates DNA synthesis in isolated nuclei b y accelerating the formation and ligation of primary pieces [4,7--9,16,17]. The possibility that the factor revealed in this study, is n o t identical with 'cytoplasmic factor' is supported by following observations. The level of activity to induce DNA synthesis in drug-treated nuclei fluctuated with phases of a cell cycle, while little fluctuation of the activity of 'cytoplasmic factor' was observed [1,11,31,32]. In addition, although drug-treated cytosol was able to stimulate DNA synthesis in S-3 h nuclei up to nearly the same level as S-3 h cytosol, the activity to induce DNA synthesis in drug-treated nuclei was found in S-3 h cytosol b u t not in drug-treated cytosol. The results shown in Table II suggest that DNA polymerase a is responsible for DNA synthesis in the complementation system. However, the observation, that even high amounts of drug-treated cytosol cannot restore DNA synthesis in drug-treated nuclei (Table I) although the cytosol contains considerable amounts of DNA polymerase ~, suggests that the complementing activity does n o t arise solely from DNA polymerase a. The fact that drug-treated cytosol supports the synthesis of high molecular weight DNA in S-3 h nuclei (Fig. 13) indicates that the cytosol contains a sufficient amount of the enzymes necessary for the step of DNA ligation, including DNA ligase. Therefore, it is unlikely that the factor in S-3 h cytosol which restores DNA synthesis in drugtreated nuclei is DNA polymerase a or DNA ligase. The fact that nuclear extract from S-3 h nuclei restored DNA synthesis in drug-treated homogenate b u t not in drug-treated nuclei indicates that the factor exists also in nuclei and that it acts in co-operating with 'cytoplasmic factor(s)'. DNA synthesis in drug-treated nuclei induced by S-3 h cytosol has been shown to require the four deoxyribonucleoside triphosphates, Mg 2÷ and ATP, and to proceed discontinuously. The fact that the DNA synthesized in the system distributed in the half heavy density region indicates that the DNA synthesis is a continuation of the synthesis at the sites where it has been already in progress in vivo. These observations seem to suggest that the DNA synthesis in the complementation system is replication type. The recovery of DNA in a shoulder of the half heavy peak toward the light density region (Fig. 3B) offers t w o possibilities, that is, initiation of new replicon and repair synthesis. Repair synthesis is generally believed n o t to require ATP. In fact, our previous observations showed that ultraviolet-irradiated repair synthesis in HeLa cell homogenate did not require ATP [29]. Therefore, it is likely that the recovery of DNA in the light density region is due to the initiation of new replicon, even though the possibility that it is due to repair synthesis requiring ATP cannot still be excluded. Although the reason w h y there is a discrepancy between the results obtained with in vivo DNA synthesis after relief of the inhibition and those with in vitro DNA synthesis in the complementation system is not clear yet, it seems to be possible that the efficiency of in vitro reconstitution of initiation system is much lower than that of elongation system. In spite of the above limitation, our system is still promising for identification and characterization of protein factors involved in eukaryotic DNA replication.
330
Acknowledgements The authors thank Dr. Susumu Ikegami for a generous gift of aphidicolin. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan. References 1 Friedman, D.L. and Mueller, G.C. (1968) Biochim. Biophys. Acta 1 6 1 , 4 5 5 - - 4 6 8 2 Lynch, W.E., Brown, R.F., Umeda, T., Langreth, S.G. and Lieberman, I. (1970) J. Biol. Chem. 245, 39 11--3916 3 Krokan, H., BjCrklid, E. and Prydz, H. (1975) Biochemistry 14, 4 2 2 7 - - 4 2 3 2 4 Franser, J.M.K. and Huberman, J.A. (1978) Biochim. Biophys. Acta 520, 271--284 5 Seki, S. and Mueller, G.C. (1976) Biochim. Biophys. Acta 4 3 5 , 2 3 6 - - 2 5 0 6 Franke, B. and Hunter, T. (1974) J. Mol. Biol. 83, 99--121 7 Tseng, B.Y. and Goulian, M. (1975) J. Mol. Biol. 9 9 , 3 1 7 - - 3 3 7 8 Franke, B. and Hunter, T. (1975) J. Virol. 15, 97--107 9 Otto, B. and Reiehard, P. (1975) J. Virol. 1 5 , 2 5 9 - - 2 6 7 10 DePamphilis, M.L. and Berg, P. (1975) J. Biol. Chem. 250, 4 3 4 8 - - 4 3 5 4 11 Brewer, E.N. (1979) Biochim. Biophys. Acta 564, 154--161 12 Seki, S., LeMahieu, M. and Mueller, G.C. (1975) Biochim. Biophys. Acta 3 7 8 , 3 3 3 - - 3 4 3 13 Berger, N.A. and J o h n s o n , E.S. (1976) Biochim. Biophys. Acta 425, 1--17 14 Hunter, T. and Franke, B. (1974) J. Mol. Biol. 8 3 , 1 2 3 - - 1 3 0 15 Tseng, B.Y. and Goulian, M. (1975) J. Mol. Biol. 9 9 , 3 3 9 - - 3 4 6 16 Kidwell, W.R. and Mueller, G.C. (1969) Biochem. Biophys. Res. C o m m u n . 3 6 , 7 5 6 - - 7 6 3 17 Krokan, H., Wist, E. and Prydz, H. (1977) Biochem. Biophys. Res. C ommun. 75, 414--419 18 MaalCe, O. and Hanawalt, P.C. (1961) J. Mol. Biol. 3 , 1 4 4 - - 1 5 5 19 Hanawalt, P.C., MaalCe, O., Cummings, D.T. and Schaechter, M. (1961) J. Mol. Biol. 3, 156--165 20 Lark, K.G. and Renger, M. (1969) J. Mol. Biol. 42, 221--235 21 Fujiwara, Y. (1972) Cancer Res. 32, 2089--2095 22 Hori, T. and Lark, K.G. (1973) J. Mol. Biol. 77, 391--404 23 Hand, R. and Tamm, I. (1972) Virology 4 7 , 3 3 1 - - 3 3 7 24 Weintraub, H. and Holtzer, H. (1972) J. Mol. Biol. 66, 13--35 25 Gautschi, J.R. and Kern, R.M. (1973) Exp. Cell Res. 80, 15--26 26 Gautschi, J.R. (1974) J. Mol. Biol. 84, 223--229 27 Stimac, E., Housman, D. and Huberman, J.A. (1977) J. Mol. Biol. 1 1 5 , 4 8 5 - - 5 1 1 28 Ono, Y., E n o m o t o , T., Hanaoka, F. and Yamada, M. (1978) Gann 6 9 , 2 0 7 - - 2 1 2 29 Kato, H., Hanaoka, F , Nishimura, T., Yamada, K. and Yamada, M. (1980) Biochim. Biophys. Acta 606, 47--56 30 Ikegami, S., Taguchi, T., Ohashi, M., Oguro, M., Nagano, H. and Mano, Y. (1978) Nature 275, 458-460 31 Reinhard, P., Maillart, P., Schluchter, M., Gautschi, J.R. and Schindler, P~. (1979) Biochim. Biophys. Aeta 564, 141--153 32 Kumar, K.V. and Friedman, D.L. (1972) Nature New Biol. 239, 74--76 33 Ono, Y., E n o m o t o , T. and Yamada, M. (1979) Gann 7 0 , 5 2 7 - - 5 3 2 34 Taylor, E.W. (1965) Exp. Cell Res. 40, 3 1 6 - - 3 3 2 35 Weiss, B.G. (1969) J. Cell Physiol. 73, 85--90