/. Biochem. 84, 145-157 (1978)
Supercoiled DNA Folded by Nonhistone Proteins in Cultured Mouse Carcinoma Cells1 Masaki NAKANE,* Toshinori IDE,** Kaijiro ANZAI,** Susumu OHARA,** and Toshiwo ANDOH**-1 •Department of Medical Chemistry, Tokyo Metropolitan Institute for Neurosciences, Musashidai, Fuchu City, Tokyo 183, and "Department of Virology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 Received for publication, January 25, 1978
Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histories could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with j--ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.
1
This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. * To whom correspondence should be addressed. Abbreviations: 4NQO, 4-nitroquinoline 1-oxide; PBS(-), 0.01 M phosphate buffer, pH7.4, 0.14M NaCI, 0.003 M KC1; SDS, sodium dodecyl sulfate. Vol. 84, No. 1, 1978
145
146
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
The DNA of mammalian cells has a total length of more than one meter, if it exists as single continuous chain of double helix. It is packed inside a cell nucleus of the order of 10 /im in diameter in a folded structure as chromatin. The DNA packing is no doubt far from random, as expected from the orderly replication and transcription in the interphase, and subsequent successful segregation of daughter chromosomes in mitosis. A considerable amount of information on the structure of chromatin has been accumulated from such studies as X-ray diffraction (1, 2), physicochemical analysis (3), electron microscopy (4, 5), and nuclease digestion (6-9). Based on these studies a new model of chromatin structure has been proposed. The model assumes that chromatin from eukaryotes comprises a linear array of spherical particles called nucleosomes or v-bodies in which DNA is supercoiled around a core of oligomers of histones (10). The apparent role of histones in stabilizing the superhelical structure of DNA in chromatin was originally established by Pardon et al. (1). Information on how the nucleosomes are organized into higher order structures is still fragmentary. The present experiments were undertaken in an attempt to elucidate this point further and to provide answers to the following specific questions. (1) Whether mammalian DNA after removal of histones by a gentle lysis procedure is still supercoiled. (2) Whether there are cellular components other than histones playing an important role in the stabilization of the supercoiling and folding of DNA, as was found in E. coli chromosomal DNA (11, 12) and as proposed for chromosomal DNA of eukaryotes (13, 14). The results described here suggest affirmative answers to these questions. Recently, we have analyzed a DNA-protein complex from cultured mouse cells and have shown that apparently SH-containing proteins other than histones were tightly bound to DNA in the complex and play an important role in maintaining the compact structure of DNA (15-17). The data obtained in the present paper are consistent with the structural model in which DNA is supercoiled and folded by a few kinds of nonhistone proteins. A brief account of this work has already been published (18, 19). Benyajati and Worcel (20), and Cook and Brazell (21) have reported similar results to ours,
in which they found supercoils of DNA in nucleoid structures isolated from cultured cells of Drosophila meJanogaster (20) and of human origin (21). One notable difference between their DNA-protein complex and ours is that their complex contained four kinds of histones and a variety of nonhistone proteins, indicating that nucleosomes are intact in their complex, whereas no histones and only a few kinds of nonhistone proteins were found in ours. MATERIALS AND METHODS Most of the materials and methods were as described previously (16-19, 22, 23). Chemicals—Pancreatic RNase A [EC 3.1.4.22] and RNase Tl [EC 3.1.4.8] were obtained, respectively, from Sigma Co. and Sankyo Co., Tokyo. Phospholipase A2 [EC 3.1.4.4] and phospholipase C [EC 3.1.4.3] purified from venom of Trimeresurus flavoviridis and culture medium of Clostridium perfringens, respectively, were kindly donated by Dr. T. Takahashi of Hoshi Pharmaceutical College, Tokyo. Pronase P [EC 3.4.24.4] was from Kaken Kagaku Co., Tokyo. 4-Nitroquinohne 1-oxide (4NQO) was a gift of Dr. Y. Kawazoe of Nagoya City University, Nagoya. Actinomycin D was from Merck, Sharp and Dohme Research Laboratories. Ethidium bromide was from Nakarai Chemicals, Tokyo. Autoradiographic emulsion NR-M2 was purchased from Konishiroku Photo Industry Co., Tokyo. Sodium dodecyl sulfate (SDS) specifically prepared for protein chemistry was purchased from Nakarai Chemicals Co., Kyoto. Cell Culture and Treatment of Cells with Various Agents—FM3A cells, originally established from C3H mouse mammary carcinoma (24), were used throughout the experiments. For treatment of cells with intercalating agents or 4NQO, cell suspension with an approximate density of 3 x 10s cells/ml was treated with the agent for 30 min at 37°C. The amount of actinomycin D bound to DNA was calculated from the radioactivity associated with DNA using [*H]actinomycin D. To label cells uniformly, we incubated cells for 20 to 24 h in growth medium with either [methyl-'HJthymidine (0.1 fiCi/ml, 14 Ci/mmol, Radiochemical Centre, L-[l,5,6-'H]fucose (10 /iCi/ml, 4.3 Ci/mmol, New England Nuclear), or [methyl-"C]choline (2 /iCi/ml, 59 mCi/mmol, Radiochemical Centre). In the case of leucine labelling, cold leucine in the /. Biochem.
SUPERCOILED, FOLDED DNA-PROTEIN COMPLEX IN MAMMALIAN CELLS
147
medium was reduced to 2 pgjml For irradiation matin was prepared from FM3A cells as described of cells with j--rays from a MCo source, a suspension by Huang and Huang (28). Chromatin was disof 3 x 106 cells/ml of medium was irradiated at solved in 6 M urea, 0.4 M guanidine hydrochloride room temperature. and 1 % 2-mercaptoethanol in 0.1 M phosphate Sucrose Density Gradient Centrifugation—The buffer, pH 7.0, and the dissociated proteins were sedimentation rates of cellular DNA in neutral separated from DNA by centrifugation at 90,000 sucrose gradients were studied as described previ- x g for 48 h. Whole chromatin proteins in the ously (16, 22). The sedimentation coefficient of supernatant were concentrated by ulrrafiltration, the DNA was calculated by using 14C-labelled and 2-mercaptoethanol was removed by passage X cl phage particles (410 S) as a reference marker. through a Sephadex G-25 column. Proteins eluted For analysis of the molecular size of denatured in the void volume were further fractionated by DNA, approximately 2 x 104 [3H]thymidine-labelled SH covalent chromatography (26). cells in 0.1 ml PBS (—) were lysed by gentle mixing Viscosity Measurements—Relative viscosity with 0.2 ml of lysis solution (0.5 N NaOH, 0.55 M was measured in a glass Ostwald-type viscometer NaCl, 0.01 M EDTA) and incubating them at immersed in a water bath kept at 36°C. Two 37°C for 1 h on top of 4.7 ml of a 5 to 20% (w/v) milliliters each of cell suspension in PBS (—) linear sucrose gradient with 0.5 N NaOH, 0.9 M (2 x 10* cells/ml) and 2% SDS solution were introNaCl, and 0.001 M EDTA. After incubation the duced into the sample chamber through fine polytubes were centrifuged in an SW 50.1 rotor (10°Q ethylene tubing at the same speed. The mixed at 36,000 rpm for 60 min. solution was incubated at 36°C for 20 min and then Large-Scale Preparation of DNA-Protein Com- the solution was allowed to drain by gravity through plex and Analysis of the Proteins by Polyacrylamide the capillary of the apparatus. The time (t) Gel Electrophorests—Approximately 6x10* cells required for the drainage was measured and the in 1.5 ml of PBS ( - ) were lysed with 3 ml of 2% relative viscosity (^rei) was calculated from the sodium dodecyl sulfate (SDS) on top of a 50 ml following equation: neutral sucrose gradient. Tubes were centrifuged J^, * in an SW 25.2 rotor (20°Q at 24,000 rpm for (1) 60 min. Fractions were collected from the bottom and those fractions which showed high viscosity where i?rei is the relative viscosity, d is the density were collected. Pooled DNA-protein complex was and t is the time required for the complete draining sonically disrupted, dialyzed against distilled water of the sample. Corresponding values with suffix and concentrated to a small volume by ultra- w are those for distilled water under the same filtration with an exclusion limit of molecular conditions. weight of 5,000. Proteins were labelled with 1MI Microscopic A utoradiography—Microscopic as described by Hunter (25). Various protein autoradiography of DNA-protein complex released samples were dissolved in sample buffer (0.063 M from cells with SDS was performed as described Tris-HCI, pH6.8, containing 2% SDS, 10% previously (19). glycerol, and 5 % 2-mercaptoethanol) and analyzed on 7.5% polyacrylamide slab gels containing RESULTS 0.375 M Tris-HCl, pH 8.8, and 0.1 % SDS. Preparation of gels and electrophoresis were performed Nature of DNA Obtained by SDS-Sucrose according to Weber et al. (27). After electroGradient Centrifugation—In order to obtain and phoresis, gels were fixed and stained with 0.25% characterize DNA in as intact a form as possible (w/v) Coomassie blue in 45 % (v/v) methanol and 1-2 xlO4 cells (0.1-0.2 fig DNA equivalent) were 9.2% (v/v) acetic acid. After destaining in a solulysed with SDS on top of 5 ml of a 5-20% linear tion of 5 % methanol and 7.5 % acetic acid, the gels sucrose gradient and centrifuged. DNA sediwere dried and brought in contact with X-ray film mented as a sharp band, as had been reported to make autoradiograms. previously (15-17, see also Fig. 5). The major Preparation of Chromatin Proteins and Frac- problems inherent in such analysis are whether the tionation by SH Covalent Chromatography—Chro-DNA thus purified still contains some cellular Vol. 84, No. 1, 1978
148
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
materials and whether the DNA behaves anomalously under the centrifugal conditions employed. The latter problem will be taken up in the next section. The former problem was examined by two different methods. (1) Labelling with radioactive precursors of various macromolecules other than DNA; (2) investigating the susceptibility of the hydrodynamic properties of the DNA to various hydrolytic enzymes. Thus, when cells were uniformly labelled with [14C]leucine, a significant radioactivity was associated with the DNA band as has been reported in a previous paper (19, see also Fig. 5). A rough estimate of the mass ratio of the protein to DNA was approximately one to five on the basis of the assumption that the specific activity of the proteins in the DNA fraction was the same as that of whole proteins of the cells. By treatment with Pronase as described in " MATERIALS AND METHODS " the label associated with the DNA band was completely eliminated. The same result was observed with 2-mercaptoethanol and with trypsin (15-17). Cells were then labelled with PH]fucose and analyzed as described. The amount of radioactivity associated with DNA fractions was less than one percent of the total phosphotungstic acid-precipitable radioactivity. When cells were labelled with ["C]choline, no radioactivity extractable with chloroform-methanol was associated with the DNA fraction. The sedimentation rate of the DNA was unaffected by RNase Tl, pancreatic RNase A, phospholipase A2 or phospholipase C suggesting that neither RNA nor phospholipids were involved, at least in such a fashion that they played a role in maintaining the faster sedimenting form of DNA, as has been suggested in E. coli chromosomes (11, 12, 29). These data suggest that the DNA obtained by the SDS-sucrose density gradient centrifugation was a DNA-protein complex essentially free of any cellular constituents. In the next experiment a large amount of the DNA-protein complex was collected by scaled-up sucrose gradient centrifugation. The content of proteins therein was still expected to be very small and therefore the proteins were radioiodinated. These proteins were mixed with chromatin proteins as a carrier and passed through a column for SHcovalent chromatography. All of the radioiodinated proteins were retained by the column and eluted with SH reagents, as expected for SHcontaining proteins. The results confirmed the
previous observation that the proteins in the complex were dissociated by 2-mercaptoethanol (17, 19). Whole chromatin proteins were electrophoresed in a 7.5% polyacrylamide slab gel and processed for autoradiography. Figure 1 shows that the 1J6I-labelled proteins from DNA-protein complex (slot C) are composed of several descrete bands predominantly in the region of molecular weights from 50,000 to 60,000. Some of them corresponded to Coomassie blue-stained bands in the SH-containing chromatin proteins (slot B) or whole chromatin proteins (slot A). Sedimentation Behavior of DNA on SDS-Sucrose Gradient Centrifugation—For the analysis of intact genomic DNA, great care must be taken to keep shear breakage to a minimum, and many workers have lysed cells on top of density gradients in the presence of various detergents, salts and enzymes. By centrifuging through the gradients -0
•60K
' "1
•52K
A B C Fig. 1. Autoradiogram after polyacrylamide gel electrophoresis of the luI-labelled proteins contained in the DNA-protein complex. Proteins in the complex were labelled with l t s I, mixed with whole chromatin proteins and fractionated by SH covalent chromatography. Whole chromatin proteins (A) and SH-containing proteins (B and C) were electrophorcsed with marker proteins (E. coli RNA polymerase (39,000, 155,000, and 165,000), bovine serum albumin (68,000), ovalbumin (43,000)) run in parallel. Gels were stained with Coomassie blue and dried, and autoradiography was performed. Coomassie blue-stained gels (A and B) and the autoradiogram (C) obtained from gel B were shown.
/. Biochem.
SUPERCOILED, FOLDED DNA-PROTEIN COMPLEX IN MAMMALIAN CELLS the DNA can be separated from other components without undue handling (30-35). In the course of these studies it was observed in support of the original results of Rubinstein and Leighton (36) that at high centrifugal force the sedimentation coefficient of large DNA is smaller than at low speeds (33, 34). Thus, as is evident in Fig. 2A, a clear dependence of s value on centrifugal force was observed with mammalian cell DNA at a concentration of 0.2 fig per gradient: 1/s decreased to a marked extent as the centrifugal force was reduced. The resulting values for s were 141S at 40,000 rpm and 256S at 5,000 rpm for the DNA obtained in the presence of 2-mercaptoethanol, which removes proteins from DNA as Pronase does (17). Figure 2B illustrates the dependence of s value on DNA concentration in the presence or absence of Pronase, i.e. DNA either as a free polynucleotide or as a DNA-protein complex exhibited concentration dependence for s values. Thus, at a speed of 30,000 rpm the values for s extrapolated to zero DNA concentration were 195S and 410S in the presence and absence of Pronase, respectively. In the following experiments 0.2 fig DNA and 20,000 rpm were used throughout. As compared with the high sedimentation rate of nucleoids obtained by lysing cells with NP40 (20), the much lower sedimentation rate of the DNAprotein complex may have been a result of disruption of the nucleoid structure under the present
l
2 3 10"4 rpm
4
experimental conditions. Another serious problem involved in the analysis of such large DNA molecules may be the entanglement of DNA molecules differing in size, resulting in a single sedimenting band. This problem is under investigation. With these reservations, we used this method for further characterization of the DNAprotein complex in the following experiments. Effect of Intercalating Agents on the Hydrodynamic Properties of DNA-Protein Complex—In order to examine whether the DNA in the complex has superhelical structure, cells were treated with various concentrations of actinomycin D and the complex was analyzed as described above. The sedimentation properties varied in a characteristic manner depending on the concentration of the agent (Fig. 3). As shown in the abscissa of Fig. 3, the amount of actinomycin D bound per unit length of DNA increased proportionately as the concentration of the drug added to a culture medium was increased up to 70 fig/ml, where approximately 40 mol of actinomycin D was bound per 10* nucleotide pairs of DNA. The minimum sedimentation rate of the complex was obtained at a concentration around 10 to 20 fgjml, i.e. 5 to 10 mol of actinomycin D bound per 104 nucleotide pairs of DNA. At doses above and below this value the sedimentation rates were larger. This pattern of transition qualitatively resembled those previously reported for supercoiled DNA from
0
0 2 0 4 0 6 0 8 1 0 DNA (^g- gradient"1)
Fig. 2. Dependence of sedimentation rate on centrifugal force and DNA concentration. Approximately 2 x 10* cells (0.2 fig DNA equivalent) were lysed with SDS in the presence of 2-mercaptoethanol and centnfuged at various speeds at 20°C (A). Various numbers of cells were lysed with SDS in the presence (O) or absence ( • ) of Pronase and centrifuged at 30,000 rpm at 20°C (B).
Vol. 84, No. 1, 1978
149
150
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
0 20 40 60 80 100 CONCENTRATION OF ACTINOMYCIN D
0
10
20
30
40
104 -MOLE OF ACTINOMYCIN D BOUND-NUCLEOTIDE PAIR"1
Fig. 3. Effects of actinomycin D and Pronase on the sedimentation of DNA-protein complex. Cells were treated with various concentrations of actinomycin D for 30 min. Washed cells (0.2 pg DNA equivalent) were then lysed with SDS on top of the gradient with ( • ) or without (O) Pronase and centnfuged at 20,000 rpm at 20°C. DNA-protein complex released was centnfuged. Cells were irradiated with r-rays at a dose of 24,000 rads from a MCo source and treated with actinomycin D before centrifugation ( A ) . The amount of actinomycin D bound per nucleotide pair of DNA was calculated from [*H]actinomycin D associated with the DNA fraction of control cells. E. coli or some plasmids thereof (11, 12, 37), but the doses which gave the minimum sedimentation coefficient of the mammalian DNA were lower than those for E. coli DNA's. Since in the present experiments the intercalation reaction was allowed to proceed in vivo where DNA exists as a nucleoprotein complex in the nucleus, the amount of actinomycin D bound to DNA should increase with time of incubation of the cells with actinomycin D added to the medium, and, if so, DNAprotein complex from these cells taken at various times of incubation should show characteristic biphasic behavior on sucrose gradient centrifugation according to the amount of bound actinomycin D. Actually, the sedimentation rate of DNA-protein complex from cells incubated with 100 ^g/ml of actinomycin D decreased after incubation for 5 min, then increased with time of incubation as shown in Fig. 4. We interpret these results as indicating that after incubation for
10 20 30 INCUBATION TIME (mm)
Fig. 4. Effect of incubation time with actinomycin D on the sedimentation of DNA-protein complex. Cells were treated with 100 pg/ml of actinomycin D for various times of incubation at 37°C, and DNA-protein complex was analyzed as described in the legend to Fig. 3.
200
A
0 200
rs
1
2000 30 0 30 0 2000
0
f 200
'
A....L1 A
o •^ 200
r i aa
0 200
1
0 200
?
30
E
0 :
I 8
30
o
0 2000 30 0 30
1
10 20 FRACTION NUMBER
Fig. 5. Stability of DNA-protein complex during actinomycin D treatment and centrifugation. Cells doubly labelled with [*H]thymidine ( • ) and ["Qleucine (O) were treated with actinomycin D at a concentration of 0 (A, B), 10 (C, D), or 100 /ig/ml (E, F). Treated and untreated control cells were lysed on top of the gradient in the presence (B, D, F) or absence (A, C, E) of Pronase and centnfuged as described in the legend to Fig. 3. J. Biochem.
SUPERCOILED, FOLDED DNA-PROTEIN COMPLEX IN MAMMALIAN CELLS
5 min, the amount of bound actinomycin D may reach 5 to lOmol per 104 nucleotide pairs to give the minimum sedimentation rate of the complex. The protein moiety of the complex was found to be tightly associated with the complex after drug treatment and subsequent analysis by sucrose density gradient centrifugation (Fig. 5). This observation is in contrast to those of Benyajati and Worcel (20), who found that as intercalation proceeded histones were removed from the nucleoid structure. Pronase treatment removed the proteins in the complex and abolished the first phase of the biphasic transition in the sedimentation rate of the intercalated complex without affecting the second phase (Figs. 3 and 5). Transitional curves similar to that with actinomycin D were obtained with other intercalating agents such as acriflavine and ethidium bromide (Fig. 6). Low doses of f-rays are known to induce single-strand breaks in DNA, so that irradiation of supercoiled DNA should lead to loss of supercoils and thus to loss of the biphasic transition of the sedimentation behavior of DNA. When cells were irradiated with /-rays prior to actinomycin D treatment the characteristic biphasic pattern was completely abolished (Fig. 3). Clearly,
0 ?0 40 60 80100 0
5 J
KF-ETHIDIUM BROMIDE ( M ) 1 2 3 4 5
0 " 10"7
10"4 10"6 1 0 ACRIFLAVIN ( M )
Fig. 6. Effects of ethidium bromide and acriflavine on the sedimentation of DNA-protcin complex. Cells were treated with various concentrations of ethidium bromide ( • ) or acnflavine (O) for 30 min at 37°C, and DNA-protein complex was analyzed as described in the legend to Fig. 3.
10
15 0 10"'
ACTINOMYCIN D O'g/ml) 10~ - j-RAY(RAD)
10"»
10" 1
4NQO ( M )
Fig. 7. Effects of actinomycin D, j--rays and 4NQO on the viscosity and sedimentation rate of DNA-protein complex. Cells were treated with actinomycin D (A), 4NQO (C), or irradiated with f-rays (B). For actinomycin treatment, treated and untreated control cells were Iysed with SDS in the presence ( • ) or absence (O) of Pronase, and the relative viscosity was measured. For 4NQO treatment and ^-irradiation, treated and untreated cells were Iysed with SDS only and the relative viscosity (O) and sedimentation rate ( x ) were measured. Vol. 84,lNo. 1, 1978
151
152
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
this is what would be expected as a result of dynamic properties of the DNA-protein complex single-strand breaks of supercoiled DNA. It was we carried out another kind of experiment in which 8 originally reported that as the sedimentation coeffi- cells were labelled with large doses of [ H]thymidine cient of DNA increases in the presence of inter- and the DNA-protein complexes after various calating agents the intrinsic viscosity decreases (38). treatments were examined by microscopic autoThis later turned out to be the case with high radiography (Fig. 9). A typical autoradiogram of molecular weight DNA (1.3 x 10s) but the opposite the control DNA-protein complex showed a comcorrelation was observed with low molecular pact mass of silver grains 80 to 100 ftm in diameter, weight DNA (106) (39, 40). This inverse relation for high molecular weight DNA was tested with 60 the mammalian DNA-protein complex. Figure 7A shows that upon intercalation of actinomycin D the relative viscosity increased initially and then § ° decreased through continued binding of actino9 30 mycin D. The relative viscosity increased when Q the complex was treated with Pronase, but a similar cr o transition was still obtained with actinomycin D. -C Effect of Strand-Scission of DNA on the > o o Hydro dynamic Properties of DNA-Protein Complex LJ —As shown in Fig. 3, the characteristic biphasic L1J transition of the sedimentation coefficient was abolished by /-irradiation. Since the main target g 0 of ionizing radiation is DNA, this could be inter£30 preted as being due to strand-scission of DNA. In this section the effect of increasing doses of 1 10 20 strand-scission inducing agents on the sedimentaFRACTION NUMBER tion coefficient and viscosity was reevaluated. Fig. 8. Effect of actinomycin D and 4NQO on the Figure 7B shows the s value and viscosity changes [single-strand scission of DNA. Cells were treated with caused by increasing doses of /-rays. The s value actinomycin D at a dose of 0(A), 10 (B) or 100/ig/ml decreased initially and leveled off, but conversely (Q, or with 4NQO at a dose of 1 x 10"' M (D) or the relative viscosity rose initially and decreased 1 X 1 0 - J M (E), and the single-stranded DNA was with further increase of dose as was seen with analyzed by alkaline sucrose gradient centrifugation as actinomycin D. Figure 7C shows a similar pattern described in " MATERIALS AND METHODS." of s value and relative viscosity changes upon treatment of cells with various doses of 4NQO, STABLE I. Effects of various treatments on the size which is a potent inducer of strand-scission of DNA distribution of the DNA-protein complex as determined (22, 41). The initial phase of rapid fall in the s by autoradiography. value and the increase in viscosity by these agents Size distribution (%) presumably reflects the loss of supercoils and the Treatment — second phase of gradual decrease in relative vis150/im cosity may reflect a gradual decrease in the molecuControl 67 33 lar weight of double-stranded DNA due to the accumulation of single-strand breaks of DNA. Pronase 72 28 This is clearly shown in Fig. 8D and 8E. This Actinomycin D (10 /ig/mT) 27 73 situation was in marked contrast to the case with Actinomycin D (10 /ig/ml) 39 61 actinomycin D, which caused little strand-scission +Pronase of DNA (Fig. 8B and 8Q. Actinomycin D (100 /
Supercoiled DNA Folded by Nonhistone Proteins in Cultured Mouse Carcinoma Cells1 Masaki NAKANE,* Toshinori IDE,** Kaijiro ANZAI,** Susumu OHARA,** and Toshiwo ANDOH**-1 •Department of Medical Chemistry, Tokyo Metropolitan Institute for Neurosciences, Musashidai, Fuchu City, Tokyo 183, and "Department of Virology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 Received for publication, January 25, 1978
Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histories could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with j--ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.
1
This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. * To whom correspondence should be addressed. Abbreviations: 4NQO, 4-nitroquinoline 1-oxide; PBS(-), 0.01 M phosphate buffer, pH7.4, 0.14M NaCI, 0.003 M KC1; SDS, sodium dodecyl sulfate. Vol. 84, No. 1, 1978
145
146
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
The DNA of mammalian cells has a total length of more than one meter, if it exists as single continuous chain of double helix. It is packed inside a cell nucleus of the order of 10 /im in diameter in a folded structure as chromatin. The DNA packing is no doubt far from random, as expected from the orderly replication and transcription in the interphase, and subsequent successful segregation of daughter chromosomes in mitosis. A considerable amount of information on the structure of chromatin has been accumulated from such studies as X-ray diffraction (1, 2), physicochemical analysis (3), electron microscopy (4, 5), and nuclease digestion (6-9). Based on these studies a new model of chromatin structure has been proposed. The model assumes that chromatin from eukaryotes comprises a linear array of spherical particles called nucleosomes or v-bodies in which DNA is supercoiled around a core of oligomers of histones (10). The apparent role of histones in stabilizing the superhelical structure of DNA in chromatin was originally established by Pardon et al. (1). Information on how the nucleosomes are organized into higher order structures is still fragmentary. The present experiments were undertaken in an attempt to elucidate this point further and to provide answers to the following specific questions. (1) Whether mammalian DNA after removal of histones by a gentle lysis procedure is still supercoiled. (2) Whether there are cellular components other than histones playing an important role in the stabilization of the supercoiling and folding of DNA, as was found in E. coli chromosomal DNA (11, 12) and as proposed for chromosomal DNA of eukaryotes (13, 14). The results described here suggest affirmative answers to these questions. Recently, we have analyzed a DNA-protein complex from cultured mouse cells and have shown that apparently SH-containing proteins other than histones were tightly bound to DNA in the complex and play an important role in maintaining the compact structure of DNA (15-17). The data obtained in the present paper are consistent with the structural model in which DNA is supercoiled and folded by a few kinds of nonhistone proteins. A brief account of this work has already been published (18, 19). Benyajati and Worcel (20), and Cook and Brazell (21) have reported similar results to ours,
in which they found supercoils of DNA in nucleoid structures isolated from cultured cells of Drosophila meJanogaster (20) and of human origin (21). One notable difference between their DNA-protein complex and ours is that their complex contained four kinds of histones and a variety of nonhistone proteins, indicating that nucleosomes are intact in their complex, whereas no histones and only a few kinds of nonhistone proteins were found in ours. MATERIALS AND METHODS Most of the materials and methods were as described previously (16-19, 22, 23). Chemicals—Pancreatic RNase A [EC 3.1.4.22] and RNase Tl [EC 3.1.4.8] were obtained, respectively, from Sigma Co. and Sankyo Co., Tokyo. Phospholipase A2 [EC 3.1.4.4] and phospholipase C [EC 3.1.4.3] purified from venom of Trimeresurus flavoviridis and culture medium of Clostridium perfringens, respectively, were kindly donated by Dr. T. Takahashi of Hoshi Pharmaceutical College, Tokyo. Pronase P [EC 3.4.24.4] was from Kaken Kagaku Co., Tokyo. 4-Nitroquinohne 1-oxide (4NQO) was a gift of Dr. Y. Kawazoe of Nagoya City University, Nagoya. Actinomycin D was from Merck, Sharp and Dohme Research Laboratories. Ethidium bromide was from Nakarai Chemicals, Tokyo. Autoradiographic emulsion NR-M2 was purchased from Konishiroku Photo Industry Co., Tokyo. Sodium dodecyl sulfate (SDS) specifically prepared for protein chemistry was purchased from Nakarai Chemicals Co., Kyoto. Cell Culture and Treatment of Cells with Various Agents—FM3A cells, originally established from C3H mouse mammary carcinoma (24), were used throughout the experiments. For treatment of cells with intercalating agents or 4NQO, cell suspension with an approximate density of 3 x 10s cells/ml was treated with the agent for 30 min at 37°C. The amount of actinomycin D bound to DNA was calculated from the radioactivity associated with DNA using [*H]actinomycin D. To label cells uniformly, we incubated cells for 20 to 24 h in growth medium with either [methyl-'HJthymidine (0.1 fiCi/ml, 14 Ci/mmol, Radiochemical Centre, L-[l,5,6-'H]fucose (10 /iCi/ml, 4.3 Ci/mmol, New England Nuclear), or [methyl-"C]choline (2 /iCi/ml, 59 mCi/mmol, Radiochemical Centre). In the case of leucine labelling, cold leucine in the /. Biochem.
SUPERCOILED, FOLDED DNA-PROTEIN COMPLEX IN MAMMALIAN CELLS
147
medium was reduced to 2 pgjml For irradiation matin was prepared from FM3A cells as described of cells with j--rays from a MCo source, a suspension by Huang and Huang (28). Chromatin was disof 3 x 106 cells/ml of medium was irradiated at solved in 6 M urea, 0.4 M guanidine hydrochloride room temperature. and 1 % 2-mercaptoethanol in 0.1 M phosphate Sucrose Density Gradient Centrifugation—The buffer, pH 7.0, and the dissociated proteins were sedimentation rates of cellular DNA in neutral separated from DNA by centrifugation at 90,000 sucrose gradients were studied as described previ- x g for 48 h. Whole chromatin proteins in the ously (16, 22). The sedimentation coefficient of supernatant were concentrated by ulrrafiltration, the DNA was calculated by using 14C-labelled and 2-mercaptoethanol was removed by passage X cl phage particles (410 S) as a reference marker. through a Sephadex G-25 column. Proteins eluted For analysis of the molecular size of denatured in the void volume were further fractionated by DNA, approximately 2 x 104 [3H]thymidine-labelled SH covalent chromatography (26). cells in 0.1 ml PBS (—) were lysed by gentle mixing Viscosity Measurements—Relative viscosity with 0.2 ml of lysis solution (0.5 N NaOH, 0.55 M was measured in a glass Ostwald-type viscometer NaCl, 0.01 M EDTA) and incubating them at immersed in a water bath kept at 36°C. Two 37°C for 1 h on top of 4.7 ml of a 5 to 20% (w/v) milliliters each of cell suspension in PBS (—) linear sucrose gradient with 0.5 N NaOH, 0.9 M (2 x 10* cells/ml) and 2% SDS solution were introNaCl, and 0.001 M EDTA. After incubation the duced into the sample chamber through fine polytubes were centrifuged in an SW 50.1 rotor (10°Q ethylene tubing at the same speed. The mixed at 36,000 rpm for 60 min. solution was incubated at 36°C for 20 min and then Large-Scale Preparation of DNA-Protein Com- the solution was allowed to drain by gravity through plex and Analysis of the Proteins by Polyacrylamide the capillary of the apparatus. The time (t) Gel Electrophorests—Approximately 6x10* cells required for the drainage was measured and the in 1.5 ml of PBS ( - ) were lysed with 3 ml of 2% relative viscosity (^rei) was calculated from the sodium dodecyl sulfate (SDS) on top of a 50 ml following equation: neutral sucrose gradient. Tubes were centrifuged J^, * in an SW 25.2 rotor (20°Q at 24,000 rpm for (1) 60 min. Fractions were collected from the bottom and those fractions which showed high viscosity where i?rei is the relative viscosity, d is the density were collected. Pooled DNA-protein complex was and t is the time required for the complete draining sonically disrupted, dialyzed against distilled water of the sample. Corresponding values with suffix and concentrated to a small volume by ultra- w are those for distilled water under the same filtration with an exclusion limit of molecular conditions. weight of 5,000. Proteins were labelled with 1MI Microscopic A utoradiography—Microscopic as described by Hunter (25). Various protein autoradiography of DNA-protein complex released samples were dissolved in sample buffer (0.063 M from cells with SDS was performed as described Tris-HCI, pH6.8, containing 2% SDS, 10% previously (19). glycerol, and 5 % 2-mercaptoethanol) and analyzed on 7.5% polyacrylamide slab gels containing RESULTS 0.375 M Tris-HCl, pH 8.8, and 0.1 % SDS. Preparation of gels and electrophoresis were performed Nature of DNA Obtained by SDS-Sucrose according to Weber et al. (27). After electroGradient Centrifugation—In order to obtain and phoresis, gels were fixed and stained with 0.25% characterize DNA in as intact a form as possible (w/v) Coomassie blue in 45 % (v/v) methanol and 1-2 xlO4 cells (0.1-0.2 fig DNA equivalent) were 9.2% (v/v) acetic acid. After destaining in a solulysed with SDS on top of 5 ml of a 5-20% linear tion of 5 % methanol and 7.5 % acetic acid, the gels sucrose gradient and centrifuged. DNA sediwere dried and brought in contact with X-ray film mented as a sharp band, as had been reported to make autoradiograms. previously (15-17, see also Fig. 5). The major Preparation of Chromatin Proteins and Frac- problems inherent in such analysis are whether the tionation by SH Covalent Chromatography—Chro-DNA thus purified still contains some cellular Vol. 84, No. 1, 1978
148
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
materials and whether the DNA behaves anomalously under the centrifugal conditions employed. The latter problem will be taken up in the next section. The former problem was examined by two different methods. (1) Labelling with radioactive precursors of various macromolecules other than DNA; (2) investigating the susceptibility of the hydrodynamic properties of the DNA to various hydrolytic enzymes. Thus, when cells were uniformly labelled with [14C]leucine, a significant radioactivity was associated with the DNA band as has been reported in a previous paper (19, see also Fig. 5). A rough estimate of the mass ratio of the protein to DNA was approximately one to five on the basis of the assumption that the specific activity of the proteins in the DNA fraction was the same as that of whole proteins of the cells. By treatment with Pronase as described in " MATERIALS AND METHODS " the label associated with the DNA band was completely eliminated. The same result was observed with 2-mercaptoethanol and with trypsin (15-17). Cells were then labelled with PH]fucose and analyzed as described. The amount of radioactivity associated with DNA fractions was less than one percent of the total phosphotungstic acid-precipitable radioactivity. When cells were labelled with ["C]choline, no radioactivity extractable with chloroform-methanol was associated with the DNA fraction. The sedimentation rate of the DNA was unaffected by RNase Tl, pancreatic RNase A, phospholipase A2 or phospholipase C suggesting that neither RNA nor phospholipids were involved, at least in such a fashion that they played a role in maintaining the faster sedimenting form of DNA, as has been suggested in E. coli chromosomes (11, 12, 29). These data suggest that the DNA obtained by the SDS-sucrose density gradient centrifugation was a DNA-protein complex essentially free of any cellular constituents. In the next experiment a large amount of the DNA-protein complex was collected by scaled-up sucrose gradient centrifugation. The content of proteins therein was still expected to be very small and therefore the proteins were radioiodinated. These proteins were mixed with chromatin proteins as a carrier and passed through a column for SHcovalent chromatography. All of the radioiodinated proteins were retained by the column and eluted with SH reagents, as expected for SHcontaining proteins. The results confirmed the
previous observation that the proteins in the complex were dissociated by 2-mercaptoethanol (17, 19). Whole chromatin proteins were electrophoresed in a 7.5% polyacrylamide slab gel and processed for autoradiography. Figure 1 shows that the 1J6I-labelled proteins from DNA-protein complex (slot C) are composed of several descrete bands predominantly in the region of molecular weights from 50,000 to 60,000. Some of them corresponded to Coomassie blue-stained bands in the SH-containing chromatin proteins (slot B) or whole chromatin proteins (slot A). Sedimentation Behavior of DNA on SDS-Sucrose Gradient Centrifugation—For the analysis of intact genomic DNA, great care must be taken to keep shear breakage to a minimum, and many workers have lysed cells on top of density gradients in the presence of various detergents, salts and enzymes. By centrifuging through the gradients -0
•60K
' "1
•52K
A B C Fig. 1. Autoradiogram after polyacrylamide gel electrophoresis of the luI-labelled proteins contained in the DNA-protein complex. Proteins in the complex were labelled with l t s I, mixed with whole chromatin proteins and fractionated by SH covalent chromatography. Whole chromatin proteins (A) and SH-containing proteins (B and C) were electrophorcsed with marker proteins (E. coli RNA polymerase (39,000, 155,000, and 165,000), bovine serum albumin (68,000), ovalbumin (43,000)) run in parallel. Gels were stained with Coomassie blue and dried, and autoradiography was performed. Coomassie blue-stained gels (A and B) and the autoradiogram (C) obtained from gel B were shown.
/. Biochem.
SUPERCOILED, FOLDED DNA-PROTEIN COMPLEX IN MAMMALIAN CELLS the DNA can be separated from other components without undue handling (30-35). In the course of these studies it was observed in support of the original results of Rubinstein and Leighton (36) that at high centrifugal force the sedimentation coefficient of large DNA is smaller than at low speeds (33, 34). Thus, as is evident in Fig. 2A, a clear dependence of s value on centrifugal force was observed with mammalian cell DNA at a concentration of 0.2 fig per gradient: 1/s decreased to a marked extent as the centrifugal force was reduced. The resulting values for s were 141S at 40,000 rpm and 256S at 5,000 rpm for the DNA obtained in the presence of 2-mercaptoethanol, which removes proteins from DNA as Pronase does (17). Figure 2B illustrates the dependence of s value on DNA concentration in the presence or absence of Pronase, i.e. DNA either as a free polynucleotide or as a DNA-protein complex exhibited concentration dependence for s values. Thus, at a speed of 30,000 rpm the values for s extrapolated to zero DNA concentration were 195S and 410S in the presence and absence of Pronase, respectively. In the following experiments 0.2 fig DNA and 20,000 rpm were used throughout. As compared with the high sedimentation rate of nucleoids obtained by lysing cells with NP40 (20), the much lower sedimentation rate of the DNAprotein complex may have been a result of disruption of the nucleoid structure under the present
l
2 3 10"4 rpm
4
experimental conditions. Another serious problem involved in the analysis of such large DNA molecules may be the entanglement of DNA molecules differing in size, resulting in a single sedimenting band. This problem is under investigation. With these reservations, we used this method for further characterization of the DNAprotein complex in the following experiments. Effect of Intercalating Agents on the Hydrodynamic Properties of DNA-Protein Complex—In order to examine whether the DNA in the complex has superhelical structure, cells were treated with various concentrations of actinomycin D and the complex was analyzed as described above. The sedimentation properties varied in a characteristic manner depending on the concentration of the agent (Fig. 3). As shown in the abscissa of Fig. 3, the amount of actinomycin D bound per unit length of DNA increased proportionately as the concentration of the drug added to a culture medium was increased up to 70 fig/ml, where approximately 40 mol of actinomycin D was bound per 10* nucleotide pairs of DNA. The minimum sedimentation rate of the complex was obtained at a concentration around 10 to 20 fgjml, i.e. 5 to 10 mol of actinomycin D bound per 104 nucleotide pairs of DNA. At doses above and below this value the sedimentation rates were larger. This pattern of transition qualitatively resembled those previously reported for supercoiled DNA from
0
0 2 0 4 0 6 0 8 1 0 DNA (^g- gradient"1)
Fig. 2. Dependence of sedimentation rate on centrifugal force and DNA concentration. Approximately 2 x 10* cells (0.2 fig DNA equivalent) were lysed with SDS in the presence of 2-mercaptoethanol and centnfuged at various speeds at 20°C (A). Various numbers of cells were lysed with SDS in the presence (O) or absence ( • ) of Pronase and centrifuged at 30,000 rpm at 20°C (B).
Vol. 84, No. 1, 1978
149
150
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
0 20 40 60 80 100 CONCENTRATION OF ACTINOMYCIN D
0
10
20
30
40
104 -MOLE OF ACTINOMYCIN D BOUND-NUCLEOTIDE PAIR"1
Fig. 3. Effects of actinomycin D and Pronase on the sedimentation of DNA-protein complex. Cells were treated with various concentrations of actinomycin D for 30 min. Washed cells (0.2 pg DNA equivalent) were then lysed with SDS on top of the gradient with ( • ) or without (O) Pronase and centnfuged at 20,000 rpm at 20°C. DNA-protein complex released was centnfuged. Cells were irradiated with r-rays at a dose of 24,000 rads from a MCo source and treated with actinomycin D before centrifugation ( A ) . The amount of actinomycin D bound per nucleotide pair of DNA was calculated from [*H]actinomycin D associated with the DNA fraction of control cells. E. coli or some plasmids thereof (11, 12, 37), but the doses which gave the minimum sedimentation coefficient of the mammalian DNA were lower than those for E. coli DNA's. Since in the present experiments the intercalation reaction was allowed to proceed in vivo where DNA exists as a nucleoprotein complex in the nucleus, the amount of actinomycin D bound to DNA should increase with time of incubation of the cells with actinomycin D added to the medium, and, if so, DNAprotein complex from these cells taken at various times of incubation should show characteristic biphasic behavior on sucrose gradient centrifugation according to the amount of bound actinomycin D. Actually, the sedimentation rate of DNA-protein complex from cells incubated with 100 ^g/ml of actinomycin D decreased after incubation for 5 min, then increased with time of incubation as shown in Fig. 4. We interpret these results as indicating that after incubation for
10 20 30 INCUBATION TIME (mm)
Fig. 4. Effect of incubation time with actinomycin D on the sedimentation of DNA-protein complex. Cells were treated with 100 pg/ml of actinomycin D for various times of incubation at 37°C, and DNA-protein complex was analyzed as described in the legend to Fig. 3.
200
A
0 200
rs
1
2000 30 0 30 0 2000
0
f 200
'
A....L1 A
o •^ 200
r i aa
0 200
1
0 200
?
30
E
0 :
I 8
30
o
0 2000 30 0 30
1
10 20 FRACTION NUMBER
Fig. 5. Stability of DNA-protein complex during actinomycin D treatment and centrifugation. Cells doubly labelled with [*H]thymidine ( • ) and ["Qleucine (O) were treated with actinomycin D at a concentration of 0 (A, B), 10 (C, D), or 100 /ig/ml (E, F). Treated and untreated control cells were lysed on top of the gradient in the presence (B, D, F) or absence (A, C, E) of Pronase and centnfuged as described in the legend to Fig. 3. J. Biochem.
SUPERCOILED, FOLDED DNA-PROTEIN COMPLEX IN MAMMALIAN CELLS
5 min, the amount of bound actinomycin D may reach 5 to lOmol per 104 nucleotide pairs to give the minimum sedimentation rate of the complex. The protein moiety of the complex was found to be tightly associated with the complex after drug treatment and subsequent analysis by sucrose density gradient centrifugation (Fig. 5). This observation is in contrast to those of Benyajati and Worcel (20), who found that as intercalation proceeded histones were removed from the nucleoid structure. Pronase treatment removed the proteins in the complex and abolished the first phase of the biphasic transition in the sedimentation rate of the intercalated complex without affecting the second phase (Figs. 3 and 5). Transitional curves similar to that with actinomycin D were obtained with other intercalating agents such as acriflavine and ethidium bromide (Fig. 6). Low doses of f-rays are known to induce single-strand breaks in DNA, so that irradiation of supercoiled DNA should lead to loss of supercoils and thus to loss of the biphasic transition of the sedimentation behavior of DNA. When cells were irradiated with /-rays prior to actinomycin D treatment the characteristic biphasic pattern was completely abolished (Fig. 3). Clearly,
0 ?0 40 60 80100 0
5 J
KF-ETHIDIUM BROMIDE ( M ) 1 2 3 4 5
0 " 10"7
10"4 10"6 1 0 ACRIFLAVIN ( M )
Fig. 6. Effects of ethidium bromide and acriflavine on the sedimentation of DNA-protcin complex. Cells were treated with various concentrations of ethidium bromide ( • ) or acnflavine (O) for 30 min at 37°C, and DNA-protein complex was analyzed as described in the legend to Fig. 3.
10
15 0 10"'
ACTINOMYCIN D O'g/ml) 10~ - j-RAY(RAD)
10"»
10" 1
4NQO ( M )
Fig. 7. Effects of actinomycin D, j--rays and 4NQO on the viscosity and sedimentation rate of DNA-protein complex. Cells were treated with actinomycin D (A), 4NQO (C), or irradiated with f-rays (B). For actinomycin treatment, treated and untreated control cells were Iysed with SDS in the presence ( • ) or absence (O) of Pronase, and the relative viscosity was measured. For 4NQO treatment and ^-irradiation, treated and untreated cells were Iysed with SDS only and the relative viscosity (O) and sedimentation rate ( x ) were measured. Vol. 84,lNo. 1, 1978
151
152
M. NAKANE, T. IDE, K. ANZAI, S. OHARA, and T. ANDOH
this is what would be expected as a result of dynamic properties of the DNA-protein complex single-strand breaks of supercoiled DNA. It was we carried out another kind of experiment in which 8 originally reported that as the sedimentation coeffi- cells were labelled with large doses of [ H]thymidine cient of DNA increases in the presence of inter- and the DNA-protein complexes after various calating agents the intrinsic viscosity decreases (38). treatments were examined by microscopic autoThis later turned out to be the case with high radiography (Fig. 9). A typical autoradiogram of molecular weight DNA (1.3 x 10s) but the opposite the control DNA-protein complex showed a comcorrelation was observed with low molecular pact mass of silver grains 80 to 100 ftm in diameter, weight DNA (106) (39, 40). This inverse relation for high molecular weight DNA was tested with 60 the mammalian DNA-protein complex. Figure 7A shows that upon intercalation of actinomycin D the relative viscosity increased initially and then § ° decreased through continued binding of actino9 30 mycin D. The relative viscosity increased when Q the complex was treated with Pronase, but a similar cr o transition was still obtained with actinomycin D. -C Effect of Strand-Scission of DNA on the > o o Hydro dynamic Properties of DNA-Protein Complex LJ —As shown in Fig. 3, the characteristic biphasic L1J transition of the sedimentation coefficient was abolished by /-irradiation. Since the main target g 0 of ionizing radiation is DNA, this could be inter£30 preted as being due to strand-scission of DNA. In this section the effect of increasing doses of 1 10 20 strand-scission inducing agents on the sedimentaFRACTION NUMBER tion coefficient and viscosity was reevaluated. Fig. 8. Effect of actinomycin D and 4NQO on the Figure 7B shows the s value and viscosity changes [single-strand scission of DNA. Cells were treated with caused by increasing doses of /-rays. The s value actinomycin D at a dose of 0(A), 10 (B) or 100/ig/ml decreased initially and leveled off, but conversely (Q, or with 4NQO at a dose of 1 x 10"' M (D) or the relative viscosity rose initially and decreased 1 X 1 0 - J M (E), and the single-stranded DNA was with further increase of dose as was seen with analyzed by alkaline sucrose gradient centrifugation as actinomycin D. Figure 7C shows a similar pattern described in " MATERIALS AND METHODS." of s value and relative viscosity changes upon treatment of cells with various doses of 4NQO, STABLE I. Effects of various treatments on the size which is a potent inducer of strand-scission of DNA distribution of the DNA-protein complex as determined (22, 41). The initial phase of rapid fall in the s by autoradiography. value and the increase in viscosity by these agents Size distribution (%) presumably reflects the loss of supercoils and the Treatment — second phase of gradual decrease in relative vis150/im cosity may reflect a gradual decrease in the molecuControl 67 33 lar weight of double-stranded DNA due to the accumulation of single-strand breaks of DNA. Pronase 72 28 This is clearly shown in Fig. 8D and 8E. This Actinomycin D (10 /ig/mT) 27 73 situation was in marked contrast to the case with Actinomycin D (10 /ig/ml) 39 61 actinomycin D, which caused little strand-scission +Pronase of DNA (Fig. 8B and 8Q. Actinomycin D (100 /
