- Email: [email protected]
Structure of nucleohistone
76
BIOCHIMICA ]ST BIOPHYSICA ACTA
BBA 95457
S T R U C T U R E OF N U C L E O H I S T O N E I. H Y D R O D Y N A M I C B E H A V I O U R
Y O S H I K I OHBA*
Department o[ Chemistry, The National Institute o[ Health o/ Japan, Shinagawaku, Tohyo (Japan) (Received October 26th, 1965)
SUMMARY
Partially dehistonized nucleohistone was prepared b y a Sephadex G-Ioo gel filtration method and its hydrodynamic properties were examined b y measuring its flow birefringence, flow dichroism and sedimentation coefficient. On association with histones the apparent length of DNA molecules decreased, and the number of base pairs perpendicular to the axis of the molecule decreased by 60 %. These configurational distortions were considered to be largely due to arginine-rich histones in the molecule. The heterogeneous components of histones were almost uniformly distributed throughout the molecular population of the nucleohistones.
INTRODUCTION
Nucleohistone can be obtained from calf-thymus glands as an aqueous solution b y dispersing chromosomal material directly in water. From measurements of the hydrodynamic and light-scattering properties of this nucleohistone, ZUBAY AND DOTY1 concluded that nucleohistone particles are less extended than free DNA molecules. The protein moiety of nucleohistone is well known to dissociate gradually in solution at an ionic strength of more than o.5. At a concentration of 2.6 M nucleohistone is generally assumed to be completely dissociated into its components, DNA and histone. The physical features of the dissociation of nucleohistone at 2.6 M NaC1 and lower ionic strengths were studied by the measurement of light scattering and viscosity 2 or of sedimentation a. The present report is on the hydrodynamic behaviour of partially deproteinized nucleohistone prepared b y a Sephadex-gel filtration method at an ionic strength of below 1.6. The methods of flow birefringence and dichroism afforded quantitative measurements of the degree of preferred folding of nucleohistone, and this is attributed to a specific histone fraction. * Present address: The F a c u l t y of P h a r m a c e u t i c a l Science, The University of Tokyo, Hongo, Tokyo, Japan.
Biochim. Biophys. ,4cta, 123 (i966) 76-83
HYDRODYNAMIC BEHAVIOUR OF NUCLEOHISTONE
77
MATERIALS AND METHODS
Preparation o[ nucleohistone, DNA and histones The nucleohistone of calf thymus was prepared b y the method of ZUBAY AND D o r y 1. The stock solution of nucleohistone (0.8 g/l) in 0.7 mM phosphate buffer (pH 6.9) was stored at 4% This preparation consisted of 48 % DNA and 52 % histones. DNA was prepared from nucleohistone according to the method of MARMUR4. Histones were extracted from calf thymus and fractionated into 3 components (JOHNS AND BUTLERS).
Chemical analyses The DNA content was determined b y an indole method e, and histone b y a Folin-phenol methodL Sometimes the concentrations of nucleohistone and DNA were determined from the absorbance at 259 m# (A250m~) assuming values of lO6 and 208 for I ~o solutions of nucleohistone and DNA, respectively. The amino acid content of histones was analyzed with an automatic amino acid analyzer (Hitachi model KLA2) according to the method of MOORE AND STEINs.
Partial dehistonization o[ nucleohistone The components of nucleohistone were partially dissociated b y adding an appropriate concentration of NaC1, in the range of o.6-1.6 M, to the nucleohistone solution. The partially dissociated mixture was then applied to a 2 cm ×30 cm Sephadex G-Ioo column, which had been equilibrated with the same salt solution, and the column was eluted with NaC1 of the same concentration at a flow rate of 0.73 ml/min. The eluate was collected in 3-ml fractions. The amounts of DNA and protein in each fraction were estimated, and fractions with the same DNA/histone ratios were combined and dialyzed against 0. 7 mM phosphate buffer.
Flow bire/ringence The extinction angle and the birefringence were measured on a Rao Instruments Birefringence Apparatus, model A-2, with white light at 2o-21 °. Measurements were usually recorded at a concentration with an A250m# of 0.8 both for DNA and nucleohistone.
Flow dichroism Flow dichroism at 259 m# was examined using a coaxial cylindrical quartz cell of the type described by WADA AND KOZAWA°. Shears up to 4500 sec -1 were accessible. A relatively high concentration of DNA and nucleohistone, corresponding to an A250ml, of 3.0-5.0, was necessary to obtain measurable dichroism.
Sedimentation A Spinco model E ultracentrifuge was used. The sedimentation coefficient was calculated according to the method of SCHUMAKER AND SCHACHMAN 10 and the Biochim. Biophys. Acta, 12 3 (1966) 76-83
78
Y. OHBA
s-integral distribution function according to the method of SHOOTER AND BUTLERll. All experiments were performed at a concentration with a n A259m/, of 0.8 both for DNA and nucleohistones, where the concentration dependence is so smalP 2 that no correction was made.
RESULTS
Partial dehistonization As the ionic strength is increased from o to o.15, the solubility of nucleohistone decreases to a few hundredths of that at zero ionic strength (ref. 13). Thereafter it increases with the ionic strength, and at above 0.5 M a transparent solution is obtained where the complex begins to dissociate. The released free histones and partially dehistonized nucleohistone were separated at an ionic strength of more than 0.6 on a column of Sephadex G-Ioo, where the dissociated histones were eluted more slowly. A typical elution pattern is shown in Fig. I. In this experiment the salt I
Protein '
['-7
3.3
3.0
-1.5
-1.0 2.C
0.2
=L
E
E
0.5 e~
1.0
o
\
0.1
0
0
0
10 Tube
15 number
20
0
Fig. I. E l u t i o n of p a r t i a l l y d e h i s t o n i z e d n u c l e o h i s t o n e f r o m a S e p h a d e x G - i o o c o l u m n (2 c m × 3o cm) in e q u i l i b r i u m w i t h o.6 M NaC1. 2 m l of o.2 % n u c l e o h i s t o n e w e re a p p l i e d a n d t h e rec o v e r y w a s 95 %. The e l u a t e w a s c o l l e c t e d in 3-ml f r a c t i o n s a t a flow r a t e of 0.73 m l / m i n . - A259 rap; . . . . A 700 m/h for e s t i m a t i o n of p r o t e i n ; - - , t h e r a t i o of p r o t e i n t o D N A , t a k e n as i . o for t h e o r i g i n a l n u c l e o h i s t o n e .
concentration was o.6 M and 2 ml of o.21 0/0 nucleohistone solution was applied. The ratio of the amount of DNA to that of protein was determined and fractions with the same ratio (Tubes No. 4-6 in Fig. I) were combined. Of the total, 25 % of the histones were dissociated at 0.6 M, and this proportion was approximately constant over the concentration range of 0.2 0/0-0.02 0/0 of nucleohistone applied. The degree of dissociation, as shown in Table I, was only dependent on the ionic strength. However, it was difficult to apply this method at a salt concentration of above 2.0 M where it was not possible to achieve good separation of the dissociated Biochim. Biophys. Acta, 123 (1966) 76 83
HYDRODYNAMIC BEHAVIOUR OF NUCLEOHISTONE TABLE
79
I
PERCENTAGE OF UNDISSOCIATED ttISTOIqE AT VARIOUS CONC1~NTRATIONS OF N a C l
Concentration o[ NaCl
Percent o[ undissociated /raction*
0.6 0.8 I.O I. 3 1.6
7o:~5 5oi 5 29±3 I7-L2 I3~2
(M)
(%)
* H i s t o n e c o n t e n t of o r i g i n a l n u c l e o h i s t o n e t a k e n as ioo.
components, possibly due to aggregation of histones 1~. A higher ionic strength was required for further deproteinization. For this purpose, CsC1 density gradient centrifugation a5 was carried out for nucleohistone, in which the ionic strength reached 5.8 and dissociated components would be separated completely on the basis of their densities. The equilibrium patterns of independent runs for DNA and for nucleohistone were completely superimposable. Moreover the DNA fraction of nucleohistone thus obtained and a preparation of DNA prepared in the conventional way were found to have identical rotary diffusion constants and flow birefringence. The ratio of lysine to arginine in the histones which remained undissociated in I.O M NaC1 was o.89 and that of whole histones was 1.3o. The undissociated histones were thus classified as arginine-rich histones (PHILLIPSI~).
Flow bire/ringence and/low dichroism In measuring birefringence, two experimental quantities were determined: the extinction angle (Z) and the amount of birefringence (An). Both were functions of the velocity gradient (G) and concentration. For molecules represented as rigid ellipsoids, Z is a function of G/O, 0 being the rotary diffusion constant of the molecule, obtained from the table of SCHERAGA, EDSALL AND GADD17for rigid prolate ellipsoids, taking the axial ratio as more than 15. Fig. 2a shows a plot of 0 against G for DNA and various preparations of nucleohistones. The values of 0 decreased with increase in the amount of histones released from nucleohistone. A decrease in the value of 0 indicates an increase in the degree of orientation of the molecules to the flow lines. The increased hydrodynamic drag can be attributed to an elongation of the molecule b y dissociation of histones, especially of less dissociable, arginine-rich ones. Calculated as a rigid ellipsoid, the apparent elongation by dehistonization reached about 1.5 times that of the original nucleohistone. To examine further the contribution of the various components of histones to the configuration of DNA, the birefringence of mixtures of DNA and various histones (very lysine-rich, slightly lysine-rich and arginine-rich) was measured at a ratio of histone to DNA of 0. 3 (Fig. 2b). The results show that even in reconstituted systems such as these, the arginine-rich histone fraction caused the greatest change in the rotary diffusion constant. The value of the flow dichroism predominantly reflects the orderliness of planar Biochim. Biophys. Acta, 123 (1966) 7 6 - 8 3
80
Y. OHBA ;9
lOOo
F 2~o 5~o 7&o ~o%o o
~6o
Velocity
gradient
~o~o
(sec - l )
Fig. 2. R o t a r y diffusion c o n s t a n t plotted against the velocity gradient, a, D N A and various preparations of nucleohistones. N u m b e r s indicate t h e protein c o n t e n t as a percentage of t h a t of t h e original nucleohistone, b, m i x t u r e s of various histones and D N A in t h e ratio of 0. 3. O - - -O, DNA; x - x , very lysine-rich fraction and DNA; O - O , slightly lysine-rich fraction and DNA; A - & , arginine-rich fraction and DNA.
purine and pyrimidine bases perpendicular to the flow-lines 9,18. The specific extinction of histone at 259 mff was only 1. 7 % of that of DNA and the contribution of histones themselves could therefore be neglected in measurement of flow dichroism. The diehroism (e//--e.)/e was measured at 259 m# where e is the molar extinction coefficient, and e// and e . the extinction coefficients for plane-polarized light, with the electric vector parallel and perpendicular to the flow-lines, respectively. The flow dichroism of preparations of nucleohistone with various amounts of histones is shown in Fig. 3. At a G value of 2000 sec -1, the dichroism of DNA was --0.26, while that of the original nucleohistone was only --0.05, which corresponded to about 2.0 % of the DNA. The disorderliness of the nucleohistone base pairs in the hydrodynamic field was largely dependent on less dissociable histones, namely arginine-rich ones. i 1200
%
./
0.2
150 x
1 ° ° q ,< I
0,1 50
,oo
do
6
Protein c o n t e n t (°/o)
Fig. 3. Flow dichroism plotted against t h e protein c o n t e n t of nucleohistone at various velocity gradients, a n d reference birefringence curve. O - O , IOOOSeC-x; Q - Q , 2ooosec-1; × - - - x , birefringence at iooo sec -1.
Biochim. Biophys. Acta, 123 (1966) 76-83
HYDRODYNAMIC BEHAVIOUR OF NUCLEOHISTONE
8I
Since the extent of orientation of DNA molecules in flow-lines was greater than that of nucleohistone molecules, the disorderliness of base pairs in flow-lines does not directly reflect the disorderliness of nucleohistone molecules. To reduce the extent of orientation of DNA, the molecules were fragmented b y a shearing force. Homogenization for I min at 70 V, though it could not be exactly defined as a shearing force, resulted in a preparation in which DNA had a rotary diffusion constant which was approximately the same as that for nucleohistone (Fig. 4). At a G value of 2000 sec -~ nucleohistone exhibited a dichroism of 38 ~o of t h a t of fragmented DNA. 0.2C
ioo
F 0,15
75 Q
...//;/
1
o.lo
× ~//o~x'~
50 o
0.05 j ~ / ./ . . . . . . . . .o ~l
o.......
........ o
~s
o '°
o/ A~'~"
~
~~ ' e " ~ I
1000
."~""~/~ I
2000
3000
Velocity grodient (sec-O
Fig. 4. Flow dichroism and rotary diffusion constant plotted against velocity gradient for DNA's and nucleohistone. - - , rotary diffusion constant; - - - , flow dichroism; A, original DNA; ×, DNA fragmented by a shearing force; O, nucleohistone.
Values for the flow birefringence (An/A2~gm~,) of preparations of nucleohistone with various amounts of histones showed a trend consistent with that obtained for flow dichroism. At a G value of 2000 sec -1 the value for the original nucleohistone corresponded to 22.7 % of that for DNA (Fig. 3), and to 47 % of that for the fragmented DNA, which had a birefringence of --53.5 × lO-8. According to STEINER AND BEERS 19, the negative birefringence of DNA can be attributed to a stacking of planar purine and pyrimidine bases perpendicular to the DNA axis. Thus the values of the birefringence are consistent with those of dichroism showing that the number of base pairs perpendicular to the molecular axis of the nucleohistone appears to be approx. 40 % of the base pairs of the fragmented DNA.
Sedimentation
B y comparing the sedimentation coefficients of partially dehistonized nucleohistones, the effect of associated histone components on the coefficient of DNA can be measured. Fig. 5 shows integral sz0,w distribution curves for original and partially dehistonized nucleohistones and for DNA. Unlike the other hydrodynamic features described above, the sedimentation
Biochim. Biophys. Acta, 123
(1966) 76-83
82
Y. OHBA
lOC
¢
g o 5C
i 15
20
i
25
30
35
S20,w
Fig. 5. Integral s-distribution function for various nucleohistones and DNA. <), original nucleohistone; ×, protein content 75 %; 0 , protein content 48 %; A, protein content 27 %; A, DNA.
coefficients of nucleohistones were changed most b y the release of intermediately dissociable histones, t h a t is slightly lysine-rich ones, rather than of arginine-rich % ones. One component of the original nucleohistone, which exhibited an sS0 20,w value of 28.o at a concentration corresponding to A2~9 m# o.8, should include only one molecule of 18.8 S DNA. 1 The s ratios of weight fractions of 80 °/o to 20 % and of 50 % to 20 % were nearly constant for original and partially dehistonized nucleohistones, showing a close similarity in the s distributions of the various nucleohistones to that of DNA. This similarity can be explained b y assuming that the various histones are almost uniformly distributed throughout the whole molecular population of the original nucleohistone.
DISCUSSION
The contribution of the various histone components to the configuration of nucleohistone was studied b y hydrodynamic analyses of a series of partially dehistonized nucleohistones in dilute solution in which nucleohistone could be dispersed into single molecules. ZUBAY AND DOTY 1 reported that nucleohistone particles are less extended than free DNA molecules. The similarities in the X - r a y diffraction patterns of DNA and nucleohistone demonstrate that the double-helical configuration of the DNA molecule is more or less well preserved in the nucleoprotein ~°. A characteristic super-helical configuration m a y be formed on association with histones, and the a p p a r e n t l e n g t h of DNA was shortened b y the association, the number of base pairs perpendicular to the direction of the axis of the molecule decreasing to 40 % of that in free DNA, largely due to arginine-rich histones. Since histones are known to be heterogeneous with regard to their amino acid composition and the ~-helix content 21, it is possible that the different histone components differ in their effect on the structure of DNA. There is much literature on the structure of mitotic chromosomes, which are generally considered to have a coiled structure. Even after telophase, some segments of chromosomes are not uncoiled, and therefore remain in a dense state. These chromosomal segments remain throughout interphase. In thymus lymphocyte, Biochir~. Biophys. Acta, 123 (1966) 76-83
HYDRODYNAMIC
BEHAVIOUR OF NUCLEOHISTONE
83
a larger part of the chromatin is in dense masses. According to LITTAU et al. 2~, this dense chromatin of thymus nuclei is formed by lysine-rich histones cross-linking the chromatin fibrils. The present experiments indicate that arginine-rich histones might be responsible for the intramolecular formation of the super-coiled structure of chromatin or mitotic chromosomes.
ACKNOWLEDGEMENTS
The author wishes to thank Dr. J. TOMIZAWA for his criticism. He is also grateful to Dr. A. WADA for measurements of flow dichroism, Dr. H. NODA and Dr. K. MARUYAMA for measurements of flow birefringence, Dr. C. NISHIMURA for CsC1 density gradient centrifugation and Dr. T. SEKIGUCHI for amino acid analyses. REFERENCES I G. ZUBAY AND P. DOTY, J. Mol. Biol., i (1959) i. 2 P. M. BAYLEY, B. N. PRESTON AND A. R. PEACOCKE, Biochim. Biophys. Acta, 55 (1962) 943. 3 G. GIANNONI AND A. R. PEACOCKE, Biochim. Biophys. Acta, 68 (1963) 157. 4 J. MARMUR, J. Mol. Biol., 3 (1961) 208. 5 E. W. JOHNS AND J. A. V. BUTLER, Biochem. J., 82 (1962) 15. 6 A. CERIOTTI, J. Biol. Chem., 198 (1952) z97. 7 0 . H. LOWRY, •. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 8 S. MOORE AND W. H. STEIN, J. Biol. Chem., 192 (1951) 663. 9 A. WADA AND S. KOZAWA, J . Polymer Sci., 2 (1964) 853. IO V. N. SCHOMAKER AND I'{. K. SCHACHMAN, Bioehim. Biophys. Acta, 23 (1957) 628. i i K. V. SHOOTER AND J. A. V. BUTLER, Trans. Farad. Soc., 52 (1956) 734. 12 J. EIGNER, C. SCHILDKRAUT AND P. DOTY, Biochim. Biophys. Acta, 55 (1962) 13. 13 A. OTH AND V. DESREUX, J. Polymer Sci., 23 (1957) 713 . 14 N. U1, Biochim. Biophys. Acta, 25 (1957) 493. 15 M. MESELSON AND F. STAHL, Proc. Natl. Acad. Sci. U.S., 47 (1958) 857. 16 D. M. P. PHILLIPS, Progr. Biophys. Biophys. Chem., 12 (1962) 211. 17 H. A. SCHERAGA, J. T. EDSALL AND J. O. GADD, J. Chem. Phys., 19 (1951) I I O I . I8 A. WADA, Biopolymers, 2 (1964) 361. 19 R. F. STEINER AND R. F. BEERS, Polynucleotides, Elsevier, A m s t e r d a m , 1961, p. 15o. 20 M. H. V. WILKINS, G. ZUBAY AND H. R. WILSON, J. Mol. Biol., I (1959) 179. 21 E. H. BRADBURY, C. CRANE-RoBINSON, D. 3][. P. PHILLIPS, E. W. JOHNS AND K. MURRAY, Nature, 2o5 (1965) 1315. 22 V. C. LITTAU, C. J. BURDICK, V. G. ALLFREY AND A. E. MILSKY, Proc. Natl. Acad. Sei. U.S., 54 (1965) 12o4.
Biochim. Biophys. Acta, 123 (1966) 76-83
BIOCHIMICA ]ST BIOPHYSICA ACTA
BBA 95457
S T R U C T U R E OF N U C L E O H I S T O N E I. H Y D R O D Y N A M I C B E H A V I O U R
Y O S H I K I OHBA*
Department o[ Chemistry, The National Institute o[ Health o/ Japan, Shinagawaku, Tohyo (Japan) (Received October 26th, 1965)
SUMMARY
Partially dehistonized nucleohistone was prepared b y a Sephadex G-Ioo gel filtration method and its hydrodynamic properties were examined b y measuring its flow birefringence, flow dichroism and sedimentation coefficient. On association with histones the apparent length of DNA molecules decreased, and the number of base pairs perpendicular to the axis of the molecule decreased by 60 %. These configurational distortions were considered to be largely due to arginine-rich histones in the molecule. The heterogeneous components of histones were almost uniformly distributed throughout the molecular population of the nucleohistones.
INTRODUCTION
Nucleohistone can be obtained from calf-thymus glands as an aqueous solution b y dispersing chromosomal material directly in water. From measurements of the hydrodynamic and light-scattering properties of this nucleohistone, ZUBAY AND DOTY1 concluded that nucleohistone particles are less extended than free DNA molecules. The protein moiety of nucleohistone is well known to dissociate gradually in solution at an ionic strength of more than o.5. At a concentration of 2.6 M nucleohistone is generally assumed to be completely dissociated into its components, DNA and histone. The physical features of the dissociation of nucleohistone at 2.6 M NaC1 and lower ionic strengths were studied by the measurement of light scattering and viscosity 2 or of sedimentation a. The present report is on the hydrodynamic behaviour of partially deproteinized nucleohistone prepared b y a Sephadex-gel filtration method at an ionic strength of below 1.6. The methods of flow birefringence and dichroism afforded quantitative measurements of the degree of preferred folding of nucleohistone, and this is attributed to a specific histone fraction. * Present address: The F a c u l t y of P h a r m a c e u t i c a l Science, The University of Tokyo, Hongo, Tokyo, Japan.
Biochim. Biophys. ,4cta, 123 (i966) 76-83
HYDRODYNAMIC BEHAVIOUR OF NUCLEOHISTONE
77
MATERIALS AND METHODS
Preparation o[ nucleohistone, DNA and histones The nucleohistone of calf thymus was prepared b y the method of ZUBAY AND D o r y 1. The stock solution of nucleohistone (0.8 g/l) in 0.7 mM phosphate buffer (pH 6.9) was stored at 4% This preparation consisted of 48 % DNA and 52 % histones. DNA was prepared from nucleohistone according to the method of MARMUR4. Histones were extracted from calf thymus and fractionated into 3 components (JOHNS AND BUTLERS).
Chemical analyses The DNA content was determined b y an indole method e, and histone b y a Folin-phenol methodL Sometimes the concentrations of nucleohistone and DNA were determined from the absorbance at 259 m# (A250m~) assuming values of lO6 and 208 for I ~o solutions of nucleohistone and DNA, respectively. The amino acid content of histones was analyzed with an automatic amino acid analyzer (Hitachi model KLA2) according to the method of MOORE AND STEINs.
Partial dehistonization o[ nucleohistone The components of nucleohistone were partially dissociated b y adding an appropriate concentration of NaC1, in the range of o.6-1.6 M, to the nucleohistone solution. The partially dissociated mixture was then applied to a 2 cm ×30 cm Sephadex G-Ioo column, which had been equilibrated with the same salt solution, and the column was eluted with NaC1 of the same concentration at a flow rate of 0.73 ml/min. The eluate was collected in 3-ml fractions. The amounts of DNA and protein in each fraction were estimated, and fractions with the same DNA/histone ratios were combined and dialyzed against 0. 7 mM phosphate buffer.
Flow bire/ringence The extinction angle and the birefringence were measured on a Rao Instruments Birefringence Apparatus, model A-2, with white light at 2o-21 °. Measurements were usually recorded at a concentration with an A250m# of 0.8 both for DNA and nucleohistone.
Flow dichroism Flow dichroism at 259 m# was examined using a coaxial cylindrical quartz cell of the type described by WADA AND KOZAWA°. Shears up to 4500 sec -1 were accessible. A relatively high concentration of DNA and nucleohistone, corresponding to an A250ml, of 3.0-5.0, was necessary to obtain measurable dichroism.
Sedimentation A Spinco model E ultracentrifuge was used. The sedimentation coefficient was calculated according to the method of SCHUMAKER AND SCHACHMAN 10 and the Biochim. Biophys. Acta, 12 3 (1966) 76-83
78
Y. OHBA
s-integral distribution function according to the method of SHOOTER AND BUTLERll. All experiments were performed at a concentration with a n A259m/, of 0.8 both for DNA and nucleohistones, where the concentration dependence is so smalP 2 that no correction was made.
RESULTS
Partial dehistonization As the ionic strength is increased from o to o.15, the solubility of nucleohistone decreases to a few hundredths of that at zero ionic strength (ref. 13). Thereafter it increases with the ionic strength, and at above 0.5 M a transparent solution is obtained where the complex begins to dissociate. The released free histones and partially dehistonized nucleohistone were separated at an ionic strength of more than 0.6 on a column of Sephadex G-Ioo, where the dissociated histones were eluted more slowly. A typical elution pattern is shown in Fig. I. In this experiment the salt I
Protein '
['-7
3.3
3.0
-1.5
-1.0 2.C
0.2
=L
E
E
0.5 e~
1.0
o
\
0.1
0
0
0
10 Tube
15 number
20
0
Fig. I. E l u t i o n of p a r t i a l l y d e h i s t o n i z e d n u c l e o h i s t o n e f r o m a S e p h a d e x G - i o o c o l u m n (2 c m × 3o cm) in e q u i l i b r i u m w i t h o.6 M NaC1. 2 m l of o.2 % n u c l e o h i s t o n e w e re a p p l i e d a n d t h e rec o v e r y w a s 95 %. The e l u a t e w a s c o l l e c t e d in 3-ml f r a c t i o n s a t a flow r a t e of 0.73 m l / m i n . - A259 rap; . . . . A 700 m/h for e s t i m a t i o n of p r o t e i n ; - - , t h e r a t i o of p r o t e i n t o D N A , t a k e n as i . o for t h e o r i g i n a l n u c l e o h i s t o n e .
concentration was o.6 M and 2 ml of o.21 0/0 nucleohistone solution was applied. The ratio of the amount of DNA to that of protein was determined and fractions with the same ratio (Tubes No. 4-6 in Fig. I) were combined. Of the total, 25 % of the histones were dissociated at 0.6 M, and this proportion was approximately constant over the concentration range of 0.2 0/0-0.02 0/0 of nucleohistone applied. The degree of dissociation, as shown in Table I, was only dependent on the ionic strength. However, it was difficult to apply this method at a salt concentration of above 2.0 M where it was not possible to achieve good separation of the dissociated Biochim. Biophys. Acta, 123 (1966) 76 83
HYDRODYNAMIC BEHAVIOUR OF NUCLEOHISTONE TABLE
79
I
PERCENTAGE OF UNDISSOCIATED ttISTOIqE AT VARIOUS CONC1~NTRATIONS OF N a C l
Concentration o[ NaCl
Percent o[ undissociated /raction*
0.6 0.8 I.O I. 3 1.6
7o:~5 5oi 5 29±3 I7-L2 I3~2
(M)
(%)
* H i s t o n e c o n t e n t of o r i g i n a l n u c l e o h i s t o n e t a k e n as ioo.
components, possibly due to aggregation of histones 1~. A higher ionic strength was required for further deproteinization. For this purpose, CsC1 density gradient centrifugation a5 was carried out for nucleohistone, in which the ionic strength reached 5.8 and dissociated components would be separated completely on the basis of their densities. The equilibrium patterns of independent runs for DNA and for nucleohistone were completely superimposable. Moreover the DNA fraction of nucleohistone thus obtained and a preparation of DNA prepared in the conventional way were found to have identical rotary diffusion constants and flow birefringence. The ratio of lysine to arginine in the histones which remained undissociated in I.O M NaC1 was o.89 and that of whole histones was 1.3o. The undissociated histones were thus classified as arginine-rich histones (PHILLIPSI~).
Flow bire/ringence and/low dichroism In measuring birefringence, two experimental quantities were determined: the extinction angle (Z) and the amount of birefringence (An). Both were functions of the velocity gradient (G) and concentration. For molecules represented as rigid ellipsoids, Z is a function of G/O, 0 being the rotary diffusion constant of the molecule, obtained from the table of SCHERAGA, EDSALL AND GADD17for rigid prolate ellipsoids, taking the axial ratio as more than 15. Fig. 2a shows a plot of 0 against G for DNA and various preparations of nucleohistones. The values of 0 decreased with increase in the amount of histones released from nucleohistone. A decrease in the value of 0 indicates an increase in the degree of orientation of the molecules to the flow lines. The increased hydrodynamic drag can be attributed to an elongation of the molecule b y dissociation of histones, especially of less dissociable, arginine-rich ones. Calculated as a rigid ellipsoid, the apparent elongation by dehistonization reached about 1.5 times that of the original nucleohistone. To examine further the contribution of the various components of histones to the configuration of DNA, the birefringence of mixtures of DNA and various histones (very lysine-rich, slightly lysine-rich and arginine-rich) was measured at a ratio of histone to DNA of 0. 3 (Fig. 2b). The results show that even in reconstituted systems such as these, the arginine-rich histone fraction caused the greatest change in the rotary diffusion constant. The value of the flow dichroism predominantly reflects the orderliness of planar Biochim. Biophys. Acta, 123 (1966) 7 6 - 8 3
80
Y. OHBA ;9
lOOo
F 2~o 5~o 7&o ~o%o o
~6o
Velocity
gradient
~o~o
(sec - l )
Fig. 2. R o t a r y diffusion c o n s t a n t plotted against the velocity gradient, a, D N A and various preparations of nucleohistones. N u m b e r s indicate t h e protein c o n t e n t as a percentage of t h a t of t h e original nucleohistone, b, m i x t u r e s of various histones and D N A in t h e ratio of 0. 3. O - - -O, DNA; x - x , very lysine-rich fraction and DNA; O - O , slightly lysine-rich fraction and DNA; A - & , arginine-rich fraction and DNA.
purine and pyrimidine bases perpendicular to the flow-lines 9,18. The specific extinction of histone at 259 mff was only 1. 7 % of that of DNA and the contribution of histones themselves could therefore be neglected in measurement of flow dichroism. The diehroism (e//--e.)/e was measured at 259 m# where e is the molar extinction coefficient, and e// and e . the extinction coefficients for plane-polarized light, with the electric vector parallel and perpendicular to the flow-lines, respectively. The flow dichroism of preparations of nucleohistone with various amounts of histones is shown in Fig. 3. At a G value of 2000 sec -1, the dichroism of DNA was --0.26, while that of the original nucleohistone was only --0.05, which corresponded to about 2.0 % of the DNA. The disorderliness of the nucleohistone base pairs in the hydrodynamic field was largely dependent on less dissociable histones, namely arginine-rich ones. i 1200
%
./
0.2
150 x
1 ° ° q ,< I
0,1 50
,oo
do
6
Protein c o n t e n t (°/o)
Fig. 3. Flow dichroism plotted against t h e protein c o n t e n t of nucleohistone at various velocity gradients, a n d reference birefringence curve. O - O , IOOOSeC-x; Q - Q , 2ooosec-1; × - - - x , birefringence at iooo sec -1.
Biochim. Biophys. Acta, 123 (1966) 76-83
HYDRODYNAMIC BEHAVIOUR OF NUCLEOHISTONE
8I
Since the extent of orientation of DNA molecules in flow-lines was greater than that of nucleohistone molecules, the disorderliness of base pairs in flow-lines does not directly reflect the disorderliness of nucleohistone molecules. To reduce the extent of orientation of DNA, the molecules were fragmented b y a shearing force. Homogenization for I min at 70 V, though it could not be exactly defined as a shearing force, resulted in a preparation in which DNA had a rotary diffusion constant which was approximately the same as that for nucleohistone (Fig. 4). At a G value of 2000 sec -~ nucleohistone exhibited a dichroism of 38 ~o of t h a t of fragmented DNA. 0.2C
ioo
F 0,15
75 Q
...//;/
1
o.lo
× ~//o~x'~
50 o
0.05 j ~ / ./ . . . . . . . . .o ~l
o.......
........ o
~s
o '°
o/ A~'~"
~
~~ ' e " ~ I
1000
."~""~/~ I
2000
3000
Velocity grodient (sec-O
Fig. 4. Flow dichroism and rotary diffusion constant plotted against velocity gradient for DNA's and nucleohistone. - - , rotary diffusion constant; - - - , flow dichroism; A, original DNA; ×, DNA fragmented by a shearing force; O, nucleohistone.
Values for the flow birefringence (An/A2~gm~,) of preparations of nucleohistone with various amounts of histones showed a trend consistent with that obtained for flow dichroism. At a G value of 2000 sec -1 the value for the original nucleohistone corresponded to 22.7 % of that for DNA (Fig. 3), and to 47 % of that for the fragmented DNA, which had a birefringence of --53.5 × lO-8. According to STEINER AND BEERS 19, the negative birefringence of DNA can be attributed to a stacking of planar purine and pyrimidine bases perpendicular to the DNA axis. Thus the values of the birefringence are consistent with those of dichroism showing that the number of base pairs perpendicular to the molecular axis of the nucleohistone appears to be approx. 40 % of the base pairs of the fragmented DNA.
Sedimentation
B y comparing the sedimentation coefficients of partially dehistonized nucleohistones, the effect of associated histone components on the coefficient of DNA can be measured. Fig. 5 shows integral sz0,w distribution curves for original and partially dehistonized nucleohistones and for DNA. Unlike the other hydrodynamic features described above, the sedimentation
Biochim. Biophys. Acta, 123
(1966) 76-83
82
Y. OHBA
lOC
¢
g o 5C
i 15
20
i
25
30
35
S20,w
Fig. 5. Integral s-distribution function for various nucleohistones and DNA. <), original nucleohistone; ×, protein content 75 %; 0 , protein content 48 %; A, protein content 27 %; A, DNA.
coefficients of nucleohistones were changed most b y the release of intermediately dissociable histones, t h a t is slightly lysine-rich ones, rather than of arginine-rich % ones. One component of the original nucleohistone, which exhibited an sS0 20,w value of 28.o at a concentration corresponding to A2~9 m# o.8, should include only one molecule of 18.8 S DNA. 1 The s ratios of weight fractions of 80 °/o to 20 % and of 50 % to 20 % were nearly constant for original and partially dehistonized nucleohistones, showing a close similarity in the s distributions of the various nucleohistones to that of DNA. This similarity can be explained b y assuming that the various histones are almost uniformly distributed throughout the whole molecular population of the original nucleohistone.
DISCUSSION
The contribution of the various histone components to the configuration of nucleohistone was studied b y hydrodynamic analyses of a series of partially dehistonized nucleohistones in dilute solution in which nucleohistone could be dispersed into single molecules. ZUBAY AND DOTY 1 reported that nucleohistone particles are less extended than free DNA molecules. The similarities in the X - r a y diffraction patterns of DNA and nucleohistone demonstrate that the double-helical configuration of the DNA molecule is more or less well preserved in the nucleoprotein ~°. A characteristic super-helical configuration m a y be formed on association with histones, and the a p p a r e n t l e n g t h of DNA was shortened b y the association, the number of base pairs perpendicular to the direction of the axis of the molecule decreasing to 40 % of that in free DNA, largely due to arginine-rich histones. Since histones are known to be heterogeneous with regard to their amino acid composition and the ~-helix content 21, it is possible that the different histone components differ in their effect on the structure of DNA. There is much literature on the structure of mitotic chromosomes, which are generally considered to have a coiled structure. Even after telophase, some segments of chromosomes are not uncoiled, and therefore remain in a dense state. These chromosomal segments remain throughout interphase. In thymus lymphocyte, Biochir~. Biophys. Acta, 123 (1966) 76-83
HYDRODYNAMIC
BEHAVIOUR OF NUCLEOHISTONE
83
a larger part of the chromatin is in dense masses. According to LITTAU et al. 2~, this dense chromatin of thymus nuclei is formed by lysine-rich histones cross-linking the chromatin fibrils. The present experiments indicate that arginine-rich histones might be responsible for the intramolecular formation of the super-coiled structure of chromatin or mitotic chromosomes.
ACKNOWLEDGEMENTS
The author wishes to thank Dr. J. TOMIZAWA for his criticism. He is also grateful to Dr. A. WADA for measurements of flow dichroism, Dr. H. NODA and Dr. K. MARUYAMA for measurements of flow birefringence, Dr. C. NISHIMURA for CsC1 density gradient centrifugation and Dr. T. SEKIGUCHI for amino acid analyses. REFERENCES I G. ZUBAY AND P. DOTY, J. Mol. Biol., i (1959) i. 2 P. M. BAYLEY, B. N. PRESTON AND A. R. PEACOCKE, Biochim. Biophys. Acta, 55 (1962) 943. 3 G. GIANNONI AND A. R. PEACOCKE, Biochim. Biophys. Acta, 68 (1963) 157. 4 J. MARMUR, J. Mol. Biol., 3 (1961) 208. 5 E. W. JOHNS AND J. A. V. BUTLER, Biochem. J., 82 (1962) 15. 6 A. CERIOTTI, J. Biol. Chem., 198 (1952) z97. 7 0 . H. LOWRY, •. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 8 S. MOORE AND W. H. STEIN, J. Biol. Chem., 192 (1951) 663. 9 A. WADA AND S. KOZAWA, J . Polymer Sci., 2 (1964) 853. IO V. N. SCHOMAKER AND I'{. K. SCHACHMAN, Bioehim. Biophys. Acta, 23 (1957) 628. i i K. V. SHOOTER AND J. A. V. BUTLER, Trans. Farad. Soc., 52 (1956) 734. 12 J. EIGNER, C. SCHILDKRAUT AND P. DOTY, Biochim. Biophys. Acta, 55 (1962) 13. 13 A. OTH AND V. DESREUX, J. Polymer Sci., 23 (1957) 713 . 14 N. U1, Biochim. Biophys. Acta, 25 (1957) 493. 15 M. MESELSON AND F. STAHL, Proc. Natl. Acad. Sci. U.S., 47 (1958) 857. 16 D. M. P. PHILLIPS, Progr. Biophys. Biophys. Chem., 12 (1962) 211. 17 H. A. SCHERAGA, J. T. EDSALL AND J. O. GADD, J. Chem. Phys., 19 (1951) I I O I . I8 A. WADA, Biopolymers, 2 (1964) 361. 19 R. F. STEINER AND R. F. BEERS, Polynucleotides, Elsevier, A m s t e r d a m , 1961, p. 15o. 20 M. H. V. WILKINS, G. ZUBAY AND H. R. WILSON, J. Mol. Biol., I (1959) 179. 21 E. H. BRADBURY, C. CRANE-RoBINSON, D. 3][. P. PHILLIPS, E. W. JOHNS AND K. MURRAY, Nature, 2o5 (1965) 1315. 22 V. C. LITTAU, C. J. BURDICK, V. G. ALLFREY AND A. E. MILSKY, Proc. Natl. Acad. Sei. U.S., 54 (1965) 12o4.
Biochim. Biophys. Acta, 123 (1966) 76-83