Replicative DNA synthesis and unscheduled DNA synthesis in permeable sarcoma cells studied by nuclease digestion

36

Biochimica et Biophysica Acta, 607 (1980) 36--42 © Elsevier/North-Holland Biomedical Press

BBA 99625

REPLICATIVE DNA SYNTHESIS AND U N S C H E D U L E D DNA SYNTHESIS IN PERMEABLE SARCOMA CELLS STUDIED BY NUCLEASE DIGESTION

SHUJI SEKI, SEKIKO WATANABE and TAKUZO ODA

Department of Biochemistry, Cancer Institute, Okayama University Medical School, 2-5-1, Shikata-cho, Okayama 700 (Japan) (Received July 5th, 1979)

Key words: DNA synthesis; Bleomycin; Nuclease digestion; Nucleosome; Chromatin replication; (Permeable sarcoma cell)

Summary A b o u t 20% of DNA replicated in vitro in permeable mouse ascites sarcoma cells showed higher sensitivity to staphylococcal nuclease than the sensitivity of bulk DNA, and the remaining part showed the same nuclease sensitivity as that o f parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in vivo in intact cells, and was close to that of DNA newly replicated in vivo in the presence of cycloheximide. Bleomycin-induced unscheduled DNA synthesis in permeable cells was highly sensitive to the nuclease. The results suggest that DNA replicated in vitro and parental nuclear protein form immature nucleosomes, probably in the same w a y as in vivo chromatin replication in the presence of protein synthesis inhibitors. It also appears that bleomycin-induced, unscheduled DNA synthesis occurs largely in the internucleosomal region.

Introduction

Eukaryotic chromatin is n o w considered to be made up of subunits called nucleosomes which consist of a b o u t 200 base-pairs of DNA and a histone octamer of H2A, H2B, H3 and H4 (for review see Ref. 1). DNA with a length of 140 base-pairs is coiled around the histone octamer and forms a nucleosome core particle. Each particle is separated b y a variable length of approx. 40 basepairs of internucleosomal DNA, that is, linker DNA, which is preferentially digestable with staphylococcal nuclease [1--4]. Chromatin replication in vivo (in intact cells) involves n o t only duplication of DNA b u t also coordinate synthesis of histories with the subsequent formation of nucleosomes. In in vitro

37 DNA synthesis using isolated nuclei or permeable cells, DNA synthesis is not associated with protein synthesis. But parental histones or nucleosomes at the replication site at least are thought to be engaged in DNA replication in vitro. We established permeable cell systems for studying DNA replication in vitro [5--7], and for studying bleomycin-induced, unscheduled DNA synthesis [8]. In this paper, to study in vitro chromatin replication, nuclease sensitivity of replicated DNA in permeable mouse ascites sarcoma cells was compared with the sensitivity of newly replicated DNA in intact cells and of repaired DNA in bleomycin-treated permeable cells. Materials and Methods

Deoxy[3H]thymidine, [3H]deoxythymidine triphosphate ([3H]dTTP) and L-[U-14C]leucine ([14C]leucine) were obtained from The Radiochemical Centre. Staphylococcal nuclease was purchased from Worthington Biochemical Corp. Cycloheximide was obtained from Sigma Chemical Co. All other chemicals were obtained as described previously [8]. DNA synthesis. Nearly exponentially growing mouse ascites sarcoma (SRC3H/He) cells were obtained from a 3
38 thymidine-incorporated intact cells were permeabilized in advance with Triton X-100/buffer B. Deoxy[3H]thymidine- or [3H]dTMP-incorporated cells were washed with 0.5 ml of Triton X-100/buffer B and then with 0.5 ml of Triton X-100/buffer C in which EDTA in Triton X-100/buffer B was replaced with 1 mM CaC12. The washed cells were suspended in 0.5 ml of Triton X-100/ buffer C supplemented with 10 units (for replicative DNA synthesis in vivo and in vitro) or 5 units (for bleomycin-induced, unscheduled DNA synthesis) of staphylococcal nuclease. The cell suspension was incubated at 37°C for 2.5 to 120 min. After incubation the suspension was chilled at 0°C and a half volume of 3M perchloric acid was added. Deoxy[3H]thymidine- or [3H]dTMPincorporated, nuclease-treated cells were collected on a glass fiber disc (GF/C). The disc was washed with 40 ml of 5% trichloroacetic acid and dried. Radioactivity of the disc was counted as described previously [6]. Polyacrylamide gel electrophoresis. DNA was prepared from nuclease-treated permeable cells ( 6 . 1 0 6 cells/tube) by the method described previously [6], and was dissolved in 0.1 ml of buffer E (40 mM Tris-HC1/20 mM sodium acetate/2 mM EDTA, pH 7.8) supplemented with 5% sucrose and 0.002% bromphenol blue. The DNA sample (0.05 ml for a tube gel or 0.03 ml for a slot of slab gels) was analyzed by two kinds of gel electrophoresis with the buffer system of Loening [9]. (1) 6% polyacrylamide tube gels (0.5 × 10.5 cm; acrylamide: N,N'-methylenebisacrylamide, 2 0 : 1 ) contained buffer E, and were electrophoresed at room temperature at 5 mA per tube for about 2.5 h. After electrophoresis, the gels were stained overnight with ethidium bromide at 1 pg/ml and photographed. (2) 6% polyacrylamide slab gels (14.5 × 12 × 0.2 cm) contained buffer E, and were electrophoresed at room temperature at 40 mA for about 6 h. After electrophoresis, the gels were stained with ethidium bromide and photographed. Then, the slab gels were impregnated with 2,5diphenyloxazol for fluorography, dried, and exposed to Kodak X-Omat R film at --70°C for 2 weeks [10]. DNA content was measured by the method of Burton [11]. Results and Discussion

Kinetics of nuclease digestion The kinetics of the digestion by staphylococcal nuclease of newly synthesized DNA and bulk DNA in permeable cells are shown in Fig. 1. The word 'bulk DNA' was used here to indicate the whole DNA measured by the method of Burton [11], but practically used as indicating parental DNA in comparison with newly synthesized DNA. Newly synthesized DNA studied in the present experiments was thought to be a minor fraction {less than 5%) of total cellular DNA. The digestability difference, as calculated from Fig. 1, at each time point between newly synthesized DNA and bulk DNA was denoted as 'rapidly digested fraction' of newly synthesized DNA compared to bulk DNA. The rapidly digested fraction of newly synthesized DNA in permeable cells was relatively large when 10 to 20% of bulk DNA became soluble, and then slightly decreased as nuclease digestion advanced further (Fig. 1). Fig. 2 shows electrophoretic patterns of DNA isolated from intact cells treated with nuclease after permeabilization (1); of DNA isolated from perme-

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Fig. I . T h e p e r c e n t a g e o f b u l k D N A o r 3 H - l a b e l e d , n e w l y s y n t h e s i z e d D N A i n p e r m e a b l e cells u n d i g e s t e d b y s t a p h y l o c o c c a l n u c l e a s e a f t e r v a r i o u s t i m e s o f i n c u b a t i o n w i t h t h e e n z y m e a t 3 7 ° C . P e r m e a b l e cells w e r e p r e p a r e d f r o m S R - C 3 H / H e cells as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . R e p l i c a t i v e D N A s y n t h e s i s ( A ) a n d b l e o m y c i n - i n d u c e d , u n s c h e d u l e d D N A s y n t h e s i s (B) w e r e c o n d u c t e d as d e s c r i b e d . [ 3 H ] d T M P i n c o r p o r a t e d cells w e r e d i g e s t e d a t 3 7 ° C f o r i n d i c a t e d t i m e s w i t h s t a p h y l o c o c c a l n u c l e a s e , a n d [ 3 H ] d T M P a n d D N A c o n t e n t in a c i d - i n s o l u b l e f r a c t i o n w e r e m e a s u r e d as d e s c r i b e d .

able cells treated with nuclease after incubation for replicative DNA synthesis (2); and of DNA isolated from permeable cells treated with nuclease after incubation for bleomycin-induced, unscheduled DNA synthesis (3). No significant difference was observed in nuclease digestion patterns of these differently

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Fig. 2. G e l e l e c t r o p h o r e s i s p a t t e r n s o f s t a p h y l o c o c c a l n u c l e a s c d i g e s t i o n p r o d u c t s . S R - C 3 H / H e cells w e r e i n c u b a t e d a t 3 7 ° C f o r 1 0 m i n f o r i n vivo r e p H e a t i v e D N A s y n t h e s i s a n d t h e n p e r m e a b i l i z e d (1); w e r e i n c u b a t e d f o r 1 0 r a i n f o r r e p l i e a t i v e D N A s y n t h e s i s a f t e r p e r m e a b i l i z a t i o n (2); o r w e r e i n c u b a t e d f o r 6 0 m i n f o r b l e o m y c i n - i n d u c e d u n s c h e d u l e d D N A s y n t h e s i s a f t e r p e r m e a b i l i z a t i o n (3). I n c u b a t e d cells (6 • 1 0 6 c e l l s / 1 . 5 m l ) w e r e t r e a t e d w i t h s t a p h y l o c o c c a l n u e l e a s e a t t h e c o n c e n t r a t i o n o f 3 0 u n i t s P e r 1 . 5 m l (1, 2) o r 1 5 u n i t s p e r 1 . 5 m l (3) a t 3 7 ° C f o r 0 m i n ( l a , 2a, 3 a ) , f o r 2 . 5 m i n ( 2 b ) , f o r 5 m i n ( I b , 2 c , 3 b ) , f o r 1 0 m i n ( l e , 2 d , 3 c ) , f o r 3 0 m i n ( l d , 2e, 3 d ) , a n d f o r 6 0 r a i n ( l e , 3 e ) . D N A p r e p a r e d f r o m n u d e a s e - t r e a t e d cells w a s e l e c t r o p h o r e s c d o n 6% P o l y a c r y l a m i d e t u b e gels as d a s c r i b e d in M a t e r i a l s a n d Methods. The Percentage of acid solubility at each point was 0 (la), 10 (lb), 19 (le), 42 (ld), 58 (le), 0 ( 2 a ) , 1 4 ( 2 b ) , 2 4 ( 2 c ) , 3 6 ( 2 d ) , 4 6 ( 2 e ) , 0 ( 3 a ) , 7 ( 3 h ) , 1 7 ( 3 c ) , 3 0 ( 3 d ) , a n d 4 0 (3e).

40 pretreated cells, though the digestability with nuclease was slightly different in preparations. This means that in vitro DNA synthesis in permeable cells or bleomycin-treatment of permeable cells did not significantly alter the basic (repeat unit} structure of chromatin. Electrophoretic patterns of DNA prepared from the cell preparations in which 10--20% of bulk DNA was digested with nuclease showed nucleosome monomer- to multimer-size DNA, but not submonomer-size DNA, indicating that the nuclease-digestion was mostly restricted within internucleosomal {linker) DNA (Fig. 2). Further digestion with nuclease resulted in the appearance of submonomer-size DNA, indicating that the digestion proceeded to nucleosome core DNA (Fig. 2).

Comparison o f rapidly digested fraction in replicative DNA synthesis and unscheduled DNA synthesis Rapidly digested fractions of newly synthesized DNA compared to bulk DNA were measured when 10--20% of bulk DNA was digested with nuclease, because it was clear from the above results that the rapidly digested fraction was derived largely from the internucleosomal region of chromatin DNA. The rapidly digested fractions measured when 10--20% of bulk DNA became soluble were almost constant and were independent of the amount of digested DNA, as indicated by little variation in the values of the rapidly digested fraction in each DNA synthesis. The rapidly digested fraction of DNA replicated in permeable cells was 22.1 + 3.0% of total replicated DNA, and the value was nearly twice that (10.7 -+ 2.5%) of DNA pulse labeled with deoxy[aH]thymidine for 10 min. The greater part {about 80%) of DNA replicated in permeable cells showed the same nuclease sensitivity as that of parental chromatin DNA. The sensitivity of bleomycin-induced, unscheduled DNA synthesis was very high (the rapidly digested fraction: 74.1-+ 0.8%) suggesting that almost all DNA synthesis occurred in the internucleosomal region. It is thought that the high nuclease-sensitivity of bleomycin-induced DNA synthesis is partly due to preferential damage of internucleosomal region of DNA by bleomycin [12] and repair of the damage. It is also possible that the constraints placed upon DNA in chromatin play a significant role in the distribution of bleomycin-induced, unscheduled DNA synthesis, as reported by Smerdon et al. [13] by using ultraviolet-induced DNA repair synthesis in cultured human diploid fibroblasts. Concerning the repair synthesis in intact cells, it has already been reported by some investigators that almost all repair-incorporated nucleotides are initially sensitive to staphylococcal nuclease and are present in the internucleosomal region of DNA [13--16]. Comparison o f nuclease sensitivity between in vitro and in vivo replicative DNA synthesis To compare nuclease sensitivity of in vitro replicative DNA synthesis with that of in vivo replicative DNA synthesis, the effect of protein synthesis inhibition on the nuclease sensitivity of newly replicated DNA was studied. When cycloheximide was added at 20 #g/0.6 ml of in vivo DNA synthesis system, DNA synthesis occurred at about half that of the cycloheximide-free control whereas [14C]leucine incorporation was almost completely inhibited (Fig. 3). Cycloheximide was without effect on replicative DNA synthesis in permeable

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Fig. 3. [ 1 4 C ] L e u c i n e i n c o r p o r a t i o n i n t o i n t a c t cells a n d p e r m e a b l e S R - C 3 H / H e cells. [ 1 4 C ] L e u c i n e ( 2 . 5 /~Ci/0.6 m l , 3 2 4 m C i / m m o l ) was a d d e d in t h e assay m i x t u r e f o r D N A s y n t h e s i s , as de~cribed in Materials a n d M e t h o d s . W h e r e a d d e d , c o n c e n t r a t i o n o f c y c l o h e x i m i d e was 20 # g / 0 . 6 m l o f r e a c t i o n m i x t u r e . I n c u b a t i o n was c o n d u c t e d a t 3 7 ° C f o r v a r i o u s t i m e s as i n d i c a t e d . [ 14 C] L e u c i n e i n c o r p o r a t e d i n t o cells w a s m e a s u 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 . T h e d a t a are e x p r e s s e d as c p m o f [ 14 C ] l e u c i n e i n c o r p o r a t e d p e r 106 cells. • e , i n t a c t cells; o o, i n t a c t cells w i t h c y c l o h e x i m i d e ; a ~, p e r m e able cells. Fig. 4. P r e s e n c e o f w i t h replicase a s s a y f o r 1 0 rain (a), 20 e l e c t r o p h o r e s i s in a

D N A r e p l i c a t e d in v i t r o in n u c l e 0 s o m e s t r u c t u r e s . P e r m e a b l e cells w e r e i n c u b a t e d m i x t u r e f o r 10 rain. S a m p l e s w e r e s u b s e q u e n t l y d i g e s t e d w i t h s t a p h y l o c o c c a l n u c l e a s e rain (13), 4 0 rain (c), or 6 0 rain (d). D N A i s o l a t e d f r o m t h e digests was s u b j e c t e d t o 6% p o l y a c r y l a m i d e slab gel a n d e x p o s e d t o film b y f l u o r o g r a p h y .

cells. No incorporation of ['4C]leucine occurred in permeable cells incubated with DNA replicase assay mixture and [ I4C]leucine. Nuclease sensitivity of newly synthesized DNA in vivo varied in accordance with the incubation time for DNA synthesis. When intact cells were incubated with deoxy[3H]thymidine for 10 min and 30 min just before harvesting, the rapidly digested fractions of newly synthesized DNA were 10.0-+ 0.5% and 1.8 -+ 1.5%, respectively. The decrease of the rapidly digested fraction with the increase in incubation time is thought to indicate that chromatin organization at or near the replication fork had returned to the organization of its nonreplicating state as reported previously [17,18]. When intact cells were incubated with deoxy[3H]thymidine in the presence of cycloheximide (20 #g/ 0.6 ml) for 10 min and 30 min, the rapidly digested fractions of newly replicated DNA were 15.1 + 1.6% and 21.8-+ 2.3%, respectively. Namely, the rapidly digested fraction increased by adding cycloheximide to the in vivo DNA synthesis mixture, as studied precisely by Weintraub [19], and approached the rapidly digested fraction of DNA replicated in permeable cells. The rapidly digested fraction of DNA replicated in permeable cells was not significantly affected by the incubation time (1 to 60 min) for DNA synthesis or by cycloheximide in the reaction mixture. Incorporation of newly replicated DNA in vitro into nucleosome core

42

regions were confirmed by fluorography (Fig. 4). Recently, Seale [20], Shelton et al. [21], and Schlaeger and Klempnauer [22] reported chromatin replication in different in vitro systems. The present results agree with their findings and suggest that DNA replication in permeable cells involves the association of the newly replicated daughter chromatids with parental histones in the formation of nucleosomes, probably as in in vivo chromatin replication with the absence of protein synthesis. So far, nuclease sensitivity of DNA synthesized even in intact cells has been studied by using isolated nuclei. There is a fear that'chromatin damage may occur in the process of nuclear isolation. To minimize chromatin damage, in the present experiment, nuclease digestion was conducted by using permeable cells. The results obtained by using permeable cells have not been so far different from those obtained by using isolated nuclei, but the present permeable cell system is thought to be useful for studying chromatin replication because of less damage to chromatin and also of easy handling of preparations. The present paper also provided a direct comparative study-system of replicative DNA synthesis and unscheduled DNA synthesis in vitro, and showed a clear difference in nuclease sensitivity between both types of DNA synthesis. Acknowledgements The authors wish to thank Mr. T. Nakamura and Ms. T. Yasui for their technical assistance, and Nippon Kayaku Co. for providing copper-free bleomycin A2. This work was supported by a grant from the Japan Ministry of Education, Science and Culture. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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