Volume 5- Number 6 June 1978 Volume 5 Number 6 June 1978
Nucleic Acids Research Research Nucleic Acids
Binding of phosphorylated histone Hi to DNA Rolf Knippers, Bemd Otto and Roswita Bohme
Fachbereich Biologie, Universit't Konstanz, D-7750 Konstanz, GFR Received 2 March 1978
ABSTRACT A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of I mole of histone HI. The DNA binding properties of this in vitro phosphorylated HI were compared with those of unmodified HI. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 2OmM-4OmM NaCl by phosphorylated HI than by unmodified Hi. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified HI are bound to DNA. It is therefore concluded that phosphorylated HI binds distributively to many or all DNA molecules available (depending on the histone/'DNA ratio) while unmodified HI binds cooperatively to a fraction of the DNA molecules in the re-
action mixture. INTRODUCTION The histone HI group differs from the other four histone species in several ways. It has a higher molecular weight (about 20,000 daltons compared to 12,000 - 15,000 daltons for the other histones). Although it is composed of about 25% lysine residues, it is more readily removed from chromatin by salt than the other histone species . The basic amino acid residues in histone HI are clustered at both ends of the primary structure2 whereas in other histones, most basic residues are located at the amino terminal end . There are several HI subfractions with interspecies and interorgan variations3. Histone HI is not a component of nucleosomes, the regular repeating units in chromatin, that are composed of two molecules each of the other four histone species. It binds independently to DNA4'5, possibly to DNA sections 6 linking adjacent nucleosomes C Information Retrieval Limited I Falconberg Court London Wl V 5FG England
2113
Nucleic Acids Research Histone HI is reversibly modified in vivo by acetylation and 8. These reactions could provide a mechanism for modulating the interaction between histones and DNA in a way that affects the structure of chromatin. It appears that phosphorylation of HI occurs at the time of DNA replication and at the beginning and during early mitosis leading to several phosphate groups per individual HI molecule
phosphorylation397
9,10,11,12,13,14 As a first approach to learn more about the functional significance of the phosphorylation reaction, we have studied the interaction of DNA with in vitro phosphorylated II and with unmodified HI using a filter binding assay and zone velocity sedimentation in sucrose gradients. The results indicate that phosphorylated histone HI binds to DNA in a distributive fashion, whereas unmodified HI binds cooperatively to DNA.
MATERIAL AND METHODS
Preparatiqn of histone HI. Histone HI was isolated from mouse Ehrlich ascites cells and from bovine lymphocytes. The preparation of histone HI has been described by Bohm, Keil and Knippers . Briefly, isolated chromatin16 was stirred for two hours in 0.1 sodium phosphate buffer (pH 7.0) containing 5 M urea, 4 M NaCl and ImM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 100,000 xg. The supernatant was adsorbed to Bio Rex 7017, and histones were eluted with 2 M NaCl. Histone HI was separated from the other histone species by carboxymethylcellulose chromatography 18
Subfractions of HI were separated by chromatography on Bio Rex 70 columns using a guanidine-HCl gradientI3. The HI preparation from mouse Ehrlich ascites cells contained one major subfraction (65-75% of the material recovered in the guanidineHC1 gradient) (Fig. 1). The HI preparation from bovine lymphocytes contained two major subfractions (peak I and II) as well as several minor species. In this study, only the first HI subfraction (pdak I) was used. Under the conditions used, no difference in the DNA binding properties between HI from 2114
Nucleic Acids Research ascites cells and HI (peak I) from lymphocytes could be detected. The experiments reported below were performed with the HI preparation from ascites cells except when 3H-labeled histone HI was used. Preparation of
3H-labeled
HI.
Bovine lymphocytes, prepared and cultivated according to Peters 19 were activated by Concanavalin A 20 At the beginning of the DNA replication phase, 1 mC (3H)-lysine (spec.activity: 33 Ci/mmol) (Amersham-Buchler) was added to a 1 1 culture. After 12 hours, the cells were harvested and radioactive histone HI was prepared as described above. The final purified HI subfraction used in this work contained in a 1.5 ml volume 1.25 mg HI with a specific radioactivity of 675 cpm//ug. Gel electrophoresis in the presence of urea21 proved the absence of modified HI (Fig. 1). ,
Phosphorylation of histone HI. The phosphorylation of HI was carried out in vitro using a chromatin associated protein kinase which preferentially phosphorylgtes histone HI2. We thus obtained a homogenous preparation of phosphorylated HI. About 0.2 mg HI (-^10 8 mole) were added to 1 ml phosphorylation buffer containing 40mM sodium phosphate, pH 7.2, I0mM magnesium sulfate, 15mM 2-mercaptoethanol, 1mM phenylmethylsulfonyl fluoride and 0.5 mg bovine serum albumin. The reaction was started by addition of (final concentration: 0.8mM; spec.act.: 1.1.x 105 -8 cpm/10 8 mole) (Amersham-Buchler) and of 50 units of chromatin bound protein kinase 22 After 2 hours at 37 C, another 50 units of protein kinase were added. Samples of 0.01 ml were removed in intervals to monitor the transfer of& _(32p) phosphate from ATP to H115. The reaction was considered to be complete when no further increase of 32P-cpm in acid precipitable material was observed (usually 4-5 hrs after the start of the reactio4 An equal volume of 8% guanidine-HCl in 0.1 M sodium phosphate (pH 7.0) was then added to the reaction mixture. Phosphorylated HI was separated from the other components of the reaction mixture by chromatography on Bio Rex 70 columns (1 cm x 3 cm) using a linear gradient from 4% to 18% guanidine HCl in 0.1 M
.-(02P)ATP
.
2115
Nucleic Acids Research sodium phosphate (pH 7.0). The peak fractions containing phosphorylated HI were extensively dialysed versus 0.1 Il sodium acetate, pH 4.2. If required, the preparation was then concentrated by precipitation with 5 volumes of acetone. The final prenaration in 0.1 Ihi sodium acetate was then divided into small aliquots and stored at -20 C until use. Each individual preparation of phosphorylated HI was not used for longer than 2-3 weeks. Preparation of DNJA.
Supercoiled Col El DNA was prepared from 2 1 cultures of the Escherichia coli strain CR54 essentially according to Cohen, Chang and I'su23. 3H-labeled Col El DNA was prepared from a I i culture labeled with I mC (3H)-thymidine (spec.act. 10 Ci//mmole) before the addition of chloramphenicol. The Col El DNA preparation was treated with proteinase K (1 mg;'ml; 4 h at 37OC) after the removal of bacterial chromosomal DNA prior to CsCl equilibrium centrifugation in the presence of ethidium bromide2'. Linear Col E1 DNA was prepared by Dr. W.Schumann from supercoiled DNA by treatment with the restriction endonuclease Eco RI which introduces one break per molecule 24 . The specific radioactivity of the Col El DNA preparations were around 20,000 cpm/,ug. DNA (spec.act.: 15,500 cpm, ug) was prepared from (>H)-thymidine labeled cultured L cells according to Otto and Knippers22. The DITA was sheared in a Virtis Model No. 00K to produce fragments with an average molecular weight of 3 x 100 (determined according to Studier2`).3H-labeled bacteriophage I4ouse
/
DTi(sptec.act. : 24,500 cpm;' ,ug) was prepared as described27 `77~~~~~~~~~~~~~~~~2
3H-labeled superhelical polyoma DNA (spec.Act.: 2800 cpm/ /ug) 28/ was prepared according to Magnusson et al. All DNA samples
were
stored at 0°C in
5mMI EDTA. DNA concentrations
were
50mFT Tris-HCl, pH 7.8,
cm determined using E21 260 nm=
20 for I mg/ml DNA. Radioactivity was determined in trichloroacetic acid precipitates collected on Whatman GF/C glass fiber
filters.
2116
Nucleic Acids Research Filter binding
assay.
Binding buffer was l0mM Tris-HCl (pH 7.6) containing lmM EDTA and 0.1 mg/ml of bovine serum albumin. DNA and HI were volumes of this buffer at the concentraI ml mixed in 0.5 tions given below. After about 10 min at room temperature the rate of about 1 mli/min through nitromixture was passed at cellulose filters (Sartorius membrane filters, SM 11 308) which had been presoaked in binding buffer. Each filter was washed twice with 5 ml binding buffer. When the binding reaction was carried out in the presence of NaCl, the wash buffer also contained NaCl at the concentration used for the binding reaction. This filter binding technique is similar to that used by others to study HI-DNA interaction29'30'31. -
a
Zone velocity sedimentation.
Linear 25% to 5% sucrose gradients were made up in binding buffer containing NaCl at the concentration required for the particular experiment. The gradients were centrifuged in the Beckman SW40 or SW41 rotor at 200C for 4 hours, if not stated otherwise, collected from the bottom and pumped through a LKBUVcord system to record the absorbance at 260 nm.
Other techniques. Protein concentrations were determined according to Schaff~29 and Weissmann, using lyophilized HI as reference. The chromatin bound cAliF independent protein kinase was prepared 22 according to Schlepper and Knippers
ner
Electrophoresis in the presence of urea and acetic acid was performed on 20 cm x 20 cm slab gels essentially according to 21 Panyim and Chalkley:. The phosphorylated amino acids in the acid hydrolysate of phosphorylated HI were determined by Dr. I. Rashed, Konstanz, using high voltage electrophoresis with unlabeled phosphoserine and phosphothreonine as markers. RESULTS In
vitro phosphorylated HI.
the specific radioactivity of the (32P)ATP used for the protein kinase reaction and from the amount of (32P)-phosphate From
2117
Nucleic Acids Research transferred to HI, we estimate that 3 moles of phosphate were bound per mole HI. This number was confirmed by the results of gel electrophoresis in the presence of urea and acetic acid2l According to Chalkley at aJL. one phosphate group reduces the mobility of HI by 1.1%. Phosphorlated HI (PH 1) migrated 3-4
Fig. I. Polyacrylamide gel electrophoresis. The urea-acetic aci+1system of was used. Panyim and Chalkley (a) Ascites cell histone HI; (b) phosphorylated ascites cell his-
tone Hl; (c) phosphorylated
7H-labeled lymphocyte HI; (d) 3H-labeled unmodified lymphocyte HI.
a
b
c
d
percent slower than unmodified HI (Fig. 1). More than 95% of the 32P-radioactivity recovered after acid hydrolySiS comigrated with phosphoserine in hij£h voltage electrophoresis (4ata not shown).
Filter binding of HI- and PHI-DNA complexes. Histone and 3H-labeled supercoiled or linear Col El DNA were mixed in a w/w ratio of 0.2 ( _ 42 moles HI/mole DNA) in 0.5 ml samples of binding buffer containing increasing NaCl concentrations. The amount of radioactive DNA retained on nitrocellulose filters is presented as a function of NaCl concentration in Fig. 2. The filter binding of histone complexes with supercoiled Col El DNA is shown in Fig. 2a. In agreement with the observations of Vogel and Singer30, we find that most radioactive DNA 2118
Nucleic Acids Research Fig. 2. Filter binding at
,w
different NaCl
con-
centrat ions.
| amples containing 1.2 u H-labeled superhelical (a) or X0-
(b)
linear
Col
El
DNA
"were made up in 0.5 ml of binding buffer with increas-
\
>
.1
ing
\
25-
\
c
salt
> b
75 4
concentrations.
To
samples was added 0 24Fug HI (*), to another set Was added the same amount of PHI (o). Filter binding was performed as described under Methods. One hundred percent corresponds to 25,500 cpm.
\one set
of
50.
1
!
20
i
ia 60
i
i i. 1.0 180 .
100
220mMNQ a
retained by HI at NaCl concentrations of about 120mM. PHI DNA complexes, however, were retained best at 20mM NaCl. Moreover, HI caused the filter binding of maximally 70% of the DNA at 120mM NaCl while more than 90% of the DNA was retained with PHI in 20mM NaCl. We show below that the fraction of filter bound DNA depends on the histone/'DNA ratio. The filter binding of histone complexes with linear 3H-labeled Col El DNA as a function of NaCl concentration is shown in Fig. 2b. Radioactive DNA was best retained at 20mM NaCl by both HI and PH1, although the fraction of filter bound material was again higher for PH1 DNA complexes. At higher NaCl concentrations, the fraction of radioactive DNA retained with HI reached a plateau and then, at 200mM NaCl and above, decreased. A plateau was not observed when PHI DNA complexes were investigated at salt concentrations of 40mM NaCl and above (Fig. 2b). was
The filter binding curve for PHI DNA complexes is rather similar for supercoiled and linear DNA, but this result is not due to nicking of supercoiled DNA by the PHI-preparation uswd, 2119
Nucleic Acids Research since sedimentation through alkaline sucrose gradients demonstrated that the DNA-strands remained covalently closed after exposure to PHI (data not shown). The fraction of filter bound DNA depends on the histone/DNA ratio. The filter binding of radioactive supercoiled Col El DNA was tested at 20mM NaCl at various ratios of HI or PHI to DNA (Fig. 3). More than 90% of the DNA was trapped on the filters at a ratio of 40-60 moles PHI/mole DNA. At a similar molar HI/DNA ratio only about 20% of the DNA was retained. Similar results were obtained when other DNA species were substituted for Col El DNA in the filter binding experiments (Table 1). These results confirm earlier shown in Fig. 2 and reports that animal virus DNA is similar to bacterial plasmid DNA in its ability to react with histone HI51. Filter binding of histone HI.
DITA does not stick to nitrocellulose filters under the conditions used in this work (Fig. 3). Free histone HI, however, does bind to filters.2934. Fig. 4 shows that a low ionic strength, somewhat less HI bound to the filters than at NaCI concentrations above 2OmM when nearly all of the (5H)-HI or Free
100
o
50-~~~~~~~~~~~~
20
40
100 60 80 moles HI/mole DNA
200
Filter binding at different histone concentrations. Fig. Oamples of I. 8ug 2H-labeled superhelical Col El DNA were placed in 0.5 fil of binding buffer with 20mM NaCl. The indicated amounts of HI (o) were added to one set of samples. A second set received the indicated amounts of PHI (o). Filter binding was performed as described. One hundred percent corresponds to 58,600 cpm. 3.
2220
Nucleic Acids Research Table 1. Filter binding of HI-DNA complexes. The experiments were carried out as described in the text for Col El DNA. Two points should: be noted: 1) the filter binding of polyoma DNA complexes with HI or with PHI is very similar to that of histone Col El DNA complexes§O. 2) under identical conditions, a larger fraction of phage T7 DNA than of sheared mouse DNA is retained on filters. This result probably ref«Rcts the preference of HI for larger over smaller DNA molecules'. ug histone ter/ug DNA
DNA
% bound to filter PHI HI
20
38
80
60
95 62
0.28
50
16
58
0.11 0.28
20
60
85
50
37
53
polyoma DNA
0.35
sheared mouse DNA
phage T7 DNA
(3H)-PHI
NaCl mM
applied to the filters was bound.
Nonradioactive supercoiled Col El DNA was added to 3H-labeled HI and PH1, respectively, before the complexes were passed through nitrocellulose filters. We found a considerable reduvtion in the 3H-labeled histones retained on filters at NaCl concentrations below 20mM (Fig. 4). This result implies that, at low ionic strength, not only HI-DNA complexes29, but also PHI-complexes are formed which do not stick to the filters. Centrifugation analysis of HI-DNA complexes. The fact that more DNA is retained on filters with PH1 than HI at 20mM NaCl (Fig. 2 and 3) could mean that PHI has a stronger affinity for DNA than does unmodified HI. The results of Fig. 2 and 3 could also mean that PHI binds to a larger fraction of the DNA molecules available. The following experiments were performed to test these possibilities.
(3H)-HI and (>H)-PHI
were mixed with unlabeled
supercoiled
Col El DNA at 20mM NaCl and centrifuged through sucrose gradients containing 20mM NaCl. The gradients were collected through a quartz cuvette to identify the position of the DNA by its absorbance at 260 nm (Fig. 5). Radioactive HI was determined in each fraction of the gradient. As shown in Fig. 5b, nearly all of the radioactive HI was 2121
Nucleic Acids Research Fig. 4. Filter binding of
(3H)-Hl.
Upper part: To 0.6 ml samples of binding buffer with the indicated NaCl concentration were added 2.8,ug (3H)-HI. The samples w6re filtered through nitrocellulose filters as describ-
I
l
_.
a ._
1I 1.
,
10
20
30 mM NoC
40
50
ed. The filter bound radioactive HI was registered (e). In a parallel experiment 8 ug unlabeled superhelical Col El DNA was added to the (5H2-Hl containing samples. Filter bound radioactivity was determined (o). Lower part: This experiment was performed exactly as the one de cribed above except that (?H)- Hl was used instead of (5H)-Hl. Filter bound radioactive histone in the absence of DNA (.), filter bound radioactive histone in the presence of unlabeled superhelical Col El DNA (o).
recoverd in those fractions of the gradient which contained the DNA. Similarily, 80% of the PHI was also associated with DNA; the remaining PHI in this experiment was found near to top of the gradient (Fig. 5c). (These experiments have been repeated at different histone/'DNA ratios from 10-100 moles histone,/,mole DNA and at salt concentrations between 2OmM-IOOmM with results essentially the same as those presented in Fig. 5). Thus HI appears to bind at least as well to DNA as PH1 does. In another set of experiments, radioactive supercoiled Col El DNA was mixed with HI or PHI in binding buffer with 20mM NaCl, both at a ratio of 1.05/ug histone/57ug DNA (,v 44 moles histone/mole DNA). The complexes were sedimented through sucrose gradients containing 20mM NaCl. Samples (0.2 ml) were removed from each fraction of the gradient and precipitated with trichloroacetic acid to determine the total radioactivity present. Another 0.2 ml sample of each fraction was diluted 1:5 in filter binding buffer containing 20mM NaCl and passed 2122
Nucleic Acids Research
Fig. 5. Sedimentation
a
ana-
lysis of histone
a2
DNA'complexes in 0.1
1000-
20mM NaCl.
b
.a
E
/
Unlabeled supercoiled Col El DNA (20 ug) in 0.4 ml binding buffer containing 2OmM NaCl (a) without histone, (b) with 4 ug (3H)-HI, and (c) with 4 u; (3H)-PH1 were centrifug4d in sucrose gradients
X
in
the
SW4I
rotor
for 36,000 rpm 4 1/4 hr. CL c ~~~~~~~~~C The relatively high background of ultraviolet light ^ material (smooth absorbing r 60. line) is due to the bovine 02 U serum albumin present in
,_--__-- -__ 5ne-_- _-__ _0 E~~~~~~~~l
% 8
at
_______aS the binding z_
200
dioactivity
slo
15
and 20
buffer. was
The
ra-
determined
in trichloroacetic acid
top nprecipitates bars).
(horizontal
frao
fraction number
through a nitrocellulose filter. The results (Fig. 6) show that, at this NaCl concentration, 32% of the DNA was retained in the presence of HI (Fig. 6a) and 72% in the presence of PHI (Fig. 6b). When 0.2 ml samples from the same fractions were diluted 1:5 into binding buffer containing 100mM NaCl, 60% of the DNA was retained by HI and 58% by PHI (Fig. 6c). This experiment has been repeated at molar ratios of histone to DNA between 20 and 80 with qualitatively similar results (data not shown). In another set of experiments, histone-DNA complexes were formed at different ratios of histone to DNA in 100mM NaCl and centrifuged through sucrose gradients containing 100mM NaCl. For example, when 5,ug 3R-labeled superhelical Col El DNA was mixed with 0.8/ug HI or PHI, centrifuged and tested for filter binding we found that with HI, 48% of the DNA was trapped on the filter and, with PHI 44% of the DNA was retained at 100mM NaCl. Filter binding of the same complexes at 20mM NaCl resulted in retention of 25% of the DNA by HI and 60% by PHI (Fig.7). The data presented in Fig. 5,6 and 7 show t)iat HI and PHI bind about equally well to DNA at 20mM and at 100mM NaCl. Nevertheless, more PHI- than HI-DNA complexes bound to filters 2123
Nucleic Acids Research
a
ll l
6
8
t. 6 0 li' g
_
4
8 1I' .4 ~~~E O .CD zz ° 2l
_ 2.
Fig. 6. Analysis of histone DNA complexes by sucrose gradient and centrifugation filter binding. (a) Complexes of Hi and radioactive suDercoiled Col El DNA were formed at 2OmM NaCl and sedimented through a sucrose gradient containing 2OmM NaCl. The SW41 rotor was used. Samples of 0.2 ml were used for tri-
|b ' \ X~~~I It s : l 2 "' ow*a~ t£, ^., \chloroacetic acid precipitation (.), for filter binding x:27 ^%s\ ff _ at 2OmH NaCl (x) and for ° filter at 100mM b NaCl (Ebinding ), respectively. o (b) Complexes of PHI and laffi 8fi fi x beled supercoiled Col El 0 Xl ~~~~~~~~C DNA were sedimented under . 1 3 identical conditions in a 6- .M1g .6 O parallel tube. Trichloroace._ l.tic acid precipitation (o) and filter binding at 2OmM 7E 4(x) T as well as at 100mM s NaCl , ,a (.A) were performed as 0. I. d above. The recovery of radiE oactive DNA was better than 2 E 42I 0
ft
_
L
.
u
I 510 15 fraction number
top
go~~~~~~~9%.
Free uncomplexed radioactive Col El DNA was centrifuged in a third parallel tube. This DNA traveled the same distance (ten fractions frm the top)as the complexes shown above (data not
shown).
at 20mM NaCl. The simplest explanation of these results is that at 20mM NaCl, PH1 is distributed on many or all (depending on the histone/DNA ratio) of the DNA molecules in the population, while binding of HI is restricted to a smaller fraction of the DNA molecules. Furthermore, unmodified HI appears to bind to a smaller fraction of the DNA molecules at 20mM than at 10imM
XaCI. It should be noted that the experiments of Fig. 6 and 7 are possible because, at the histone/DNA ratio chosen, the sedimentation rate of histone DNA complexes (Fig. 5b and c) did not differ much from that of free DNA (Fig. 5a). (Free DNA was always sedimented as a control in a parallel tube when experi2124
,^;* /1 analyzed as described in the U l a olegend to Fig. 6 except that Z 100mM NaCl was used in the \ i binding buffer and in the su.4.4 |Al crose gradient. After sucroC se gradient centrifugation of the HI-Col El DNA com.2plexes aliquots assayed _wI |q*^ by trichloroacetic acid pre-
o
x
were
,C_..D_*_
20mM NaCl
b
*8-
R CDR
.
.\, t
x 8 6
._ E "
cipitation (e), filter binding at 100mM NaCl (A4) and
c
centrifuged through.
sucrose
gradients containing 100mM NaCl. Aliquots were assayed by
@
(x).
(b) PHI-DNA complexes were formed at 100mM NaCl and
acid
precipitation
(o)
and filter binding at 100mM (A) and 20mM (x) NaCl.. l lThe recovery of radioactive DNA was about 90%. E
4--
2.-~~~~~~~~~~~~a 5
0l 15 fraction number
top
ments of the type shown in Fig. 6 and 7 were performed. At the histone/DNA ratios used in these experiments, no difference in sedimentation rate between free and complexed DNA was observed This analysis, of course, was not sensitive enough to detect possible small differences). This result is surprising since a cooperative binding of HI ot DNA at a w,w ratio of, for example, 0.4 caused filter binding of about 30% of the DNA present (Fig. 3). This fraction should contain DNA molecules complexed with somewhat more than an equal mass of histones. Since this mass increase did not cause an enhanded sedimentation rate we tentatively conclude that the frictional coefficient of the complexes must be higher than that of free DNA, possibly due to a reduction of the degree of superhelicity or to an alteration of the structure of the superhelical DNA (interwound superhelix to toroidal superhelix or vice versa). These complications may be avoided by studying the sedimen2125
Nucleic Acids Research tation of linear Col El DNA-histone complexes. Mixtures of linear 3H-labeled Col El DNA and either or PHI in binding buffer containing 20mM NaCl were centrifuged through sucrose density gradients as described above for the experiments of Fig. 6 and Fig. 7 (Fig. 8). In contrast to the results with supercoiled DNA, the peak fraction of the HI complexes with linear DNA sedimented about 1.3 times faster than free DNA. The PHI-DNA complexes sedimented in a sharp peak about 1.1 times faster than free DNA. On the average 47% of all recovered DNA was retained on nitrocellulose filters by HI and 75% by PH1, respectively. When the filter binding of individual fractions of the gradients was tested, we found that almost 70% of the fastest sedimenting fraction of the HI-DNA complexes was retained but less of the slower sedimenting material bound to filters (Fig. 8a). How-
HI
la
1004
e5
50
c
Fig. 8. Sucrose gradient of histone aanalysis complexes with linear Col El DNA. (a) As a control, linear 3Elabeled Col El DNA was cen-
-*
4
6
A_
.
trifuged through
a sucrose
gradient containing 20mM NaCl
(.).
Complexes of
.2 ug of linear labeled DNA with 0.4 ug of HI were sedimented ih a parallel tube(\). I
.'x
e"
l
l
X !t\Acid precipitable radioactiv2 *
Nucleic Acids Research Research Nucleic Acids
Binding of phosphorylated histone Hi to DNA Rolf Knippers, Bemd Otto and Roswita Bohme
Fachbereich Biologie, Universit't Konstanz, D-7750 Konstanz, GFR Received 2 March 1978
ABSTRACT A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of I mole of histone HI. The DNA binding properties of this in vitro phosphorylated HI were compared with those of unmodified HI. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 2OmM-4OmM NaCl by phosphorylated HI than by unmodified Hi. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified HI are bound to DNA. It is therefore concluded that phosphorylated HI binds distributively to many or all DNA molecules available (depending on the histone/'DNA ratio) while unmodified HI binds cooperatively to a fraction of the DNA molecules in the re-
action mixture. INTRODUCTION The histone HI group differs from the other four histone species in several ways. It has a higher molecular weight (about 20,000 daltons compared to 12,000 - 15,000 daltons for the other histones). Although it is composed of about 25% lysine residues, it is more readily removed from chromatin by salt than the other histone species . The basic amino acid residues in histone HI are clustered at both ends of the primary structure2 whereas in other histones, most basic residues are located at the amino terminal end . There are several HI subfractions with interspecies and interorgan variations3. Histone HI is not a component of nucleosomes, the regular repeating units in chromatin, that are composed of two molecules each of the other four histone species. It binds independently to DNA4'5, possibly to DNA sections 6 linking adjacent nucleosomes C Information Retrieval Limited I Falconberg Court London Wl V 5FG England
2113
Nucleic Acids Research Histone HI is reversibly modified in vivo by acetylation and 8. These reactions could provide a mechanism for modulating the interaction between histones and DNA in a way that affects the structure of chromatin. It appears that phosphorylation of HI occurs at the time of DNA replication and at the beginning and during early mitosis leading to several phosphate groups per individual HI molecule
phosphorylation397
9,10,11,12,13,14 As a first approach to learn more about the functional significance of the phosphorylation reaction, we have studied the interaction of DNA with in vitro phosphorylated II and with unmodified HI using a filter binding assay and zone velocity sedimentation in sucrose gradients. The results indicate that phosphorylated histone HI binds to DNA in a distributive fashion, whereas unmodified HI binds cooperatively to DNA.
MATERIAL AND METHODS
Preparatiqn of histone HI. Histone HI was isolated from mouse Ehrlich ascites cells and from bovine lymphocytes. The preparation of histone HI has been described by Bohm, Keil and Knippers . Briefly, isolated chromatin16 was stirred for two hours in 0.1 sodium phosphate buffer (pH 7.0) containing 5 M urea, 4 M NaCl and ImM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 100,000 xg. The supernatant was adsorbed to Bio Rex 7017, and histones were eluted with 2 M NaCl. Histone HI was separated from the other histone species by carboxymethylcellulose chromatography 18
Subfractions of HI were separated by chromatography on Bio Rex 70 columns using a guanidine-HCl gradientI3. The HI preparation from mouse Ehrlich ascites cells contained one major subfraction (65-75% of the material recovered in the guanidineHC1 gradient) (Fig. 1). The HI preparation from bovine lymphocytes contained two major subfractions (peak I and II) as well as several minor species. In this study, only the first HI subfraction (pdak I) was used. Under the conditions used, no difference in the DNA binding properties between HI from 2114
Nucleic Acids Research ascites cells and HI (peak I) from lymphocytes could be detected. The experiments reported below were performed with the HI preparation from ascites cells except when 3H-labeled histone HI was used. Preparation of
3H-labeled
HI.
Bovine lymphocytes, prepared and cultivated according to Peters 19 were activated by Concanavalin A 20 At the beginning of the DNA replication phase, 1 mC (3H)-lysine (spec.activity: 33 Ci/mmol) (Amersham-Buchler) was added to a 1 1 culture. After 12 hours, the cells were harvested and radioactive histone HI was prepared as described above. The final purified HI subfraction used in this work contained in a 1.5 ml volume 1.25 mg HI with a specific radioactivity of 675 cpm//ug. Gel electrophoresis in the presence of urea21 proved the absence of modified HI (Fig. 1). ,
Phosphorylation of histone HI. The phosphorylation of HI was carried out in vitro using a chromatin associated protein kinase which preferentially phosphorylgtes histone HI2. We thus obtained a homogenous preparation of phosphorylated HI. About 0.2 mg HI (-^10 8 mole) were added to 1 ml phosphorylation buffer containing 40mM sodium phosphate, pH 7.2, I0mM magnesium sulfate, 15mM 2-mercaptoethanol, 1mM phenylmethylsulfonyl fluoride and 0.5 mg bovine serum albumin. The reaction was started by addition of (final concentration: 0.8mM; spec.act.: 1.1.x 105 -8 cpm/10 8 mole) (Amersham-Buchler) and of 50 units of chromatin bound protein kinase 22 After 2 hours at 37 C, another 50 units of protein kinase were added. Samples of 0.01 ml were removed in intervals to monitor the transfer of& _(32p) phosphate from ATP to H115. The reaction was considered to be complete when no further increase of 32P-cpm in acid precipitable material was observed (usually 4-5 hrs after the start of the reactio4 An equal volume of 8% guanidine-HCl in 0.1 M sodium phosphate (pH 7.0) was then added to the reaction mixture. Phosphorylated HI was separated from the other components of the reaction mixture by chromatography on Bio Rex 70 columns (1 cm x 3 cm) using a linear gradient from 4% to 18% guanidine HCl in 0.1 M
.-(02P)ATP
.
2115
Nucleic Acids Research sodium phosphate (pH 7.0). The peak fractions containing phosphorylated HI were extensively dialysed versus 0.1 Il sodium acetate, pH 4.2. If required, the preparation was then concentrated by precipitation with 5 volumes of acetone. The final prenaration in 0.1 Ihi sodium acetate was then divided into small aliquots and stored at -20 C until use. Each individual preparation of phosphorylated HI was not used for longer than 2-3 weeks. Preparation of DNJA.
Supercoiled Col El DNA was prepared from 2 1 cultures of the Escherichia coli strain CR54 essentially according to Cohen, Chang and I'su23. 3H-labeled Col El DNA was prepared from a I i culture labeled with I mC (3H)-thymidine (spec.act. 10 Ci//mmole) before the addition of chloramphenicol. The Col El DNA preparation was treated with proteinase K (1 mg;'ml; 4 h at 37OC) after the removal of bacterial chromosomal DNA prior to CsCl equilibrium centrifugation in the presence of ethidium bromide2'. Linear Col E1 DNA was prepared by Dr. W.Schumann from supercoiled DNA by treatment with the restriction endonuclease Eco RI which introduces one break per molecule 24 . The specific radioactivity of the Col El DNA preparations were around 20,000 cpm/,ug. DNA (spec.act.: 15,500 cpm, ug) was prepared from (>H)-thymidine labeled cultured L cells according to Otto and Knippers22. The DITA was sheared in a Virtis Model No. 00K to produce fragments with an average molecular weight of 3 x 100 (determined according to Studier2`).3H-labeled bacteriophage I4ouse
/
DTi(sptec.act. : 24,500 cpm;' ,ug) was prepared as described27 `77~~~~~~~~~~~~~~~~2
3H-labeled superhelical polyoma DNA (spec.Act.: 2800 cpm/ /ug) 28/ was prepared according to Magnusson et al. All DNA samples
were
stored at 0°C in
5mMI EDTA. DNA concentrations
were
50mFT Tris-HCl, pH 7.8,
cm determined using E21 260 nm=
20 for I mg/ml DNA. Radioactivity was determined in trichloroacetic acid precipitates collected on Whatman GF/C glass fiber
filters.
2116
Nucleic Acids Research Filter binding
assay.
Binding buffer was l0mM Tris-HCl (pH 7.6) containing lmM EDTA and 0.1 mg/ml of bovine serum albumin. DNA and HI were volumes of this buffer at the concentraI ml mixed in 0.5 tions given below. After about 10 min at room temperature the rate of about 1 mli/min through nitromixture was passed at cellulose filters (Sartorius membrane filters, SM 11 308) which had been presoaked in binding buffer. Each filter was washed twice with 5 ml binding buffer. When the binding reaction was carried out in the presence of NaCl, the wash buffer also contained NaCl at the concentration used for the binding reaction. This filter binding technique is similar to that used by others to study HI-DNA interaction29'30'31. -
a
Zone velocity sedimentation.
Linear 25% to 5% sucrose gradients were made up in binding buffer containing NaCl at the concentration required for the particular experiment. The gradients were centrifuged in the Beckman SW40 or SW41 rotor at 200C for 4 hours, if not stated otherwise, collected from the bottom and pumped through a LKBUVcord system to record the absorbance at 260 nm.
Other techniques. Protein concentrations were determined according to Schaff~29 and Weissmann, using lyophilized HI as reference. The chromatin bound cAliF independent protein kinase was prepared 22 according to Schlepper and Knippers
ner
Electrophoresis in the presence of urea and acetic acid was performed on 20 cm x 20 cm slab gels essentially according to 21 Panyim and Chalkley:. The phosphorylated amino acids in the acid hydrolysate of phosphorylated HI were determined by Dr. I. Rashed, Konstanz, using high voltage electrophoresis with unlabeled phosphoserine and phosphothreonine as markers. RESULTS In
vitro phosphorylated HI.
the specific radioactivity of the (32P)ATP used for the protein kinase reaction and from the amount of (32P)-phosphate From
2117
Nucleic Acids Research transferred to HI, we estimate that 3 moles of phosphate were bound per mole HI. This number was confirmed by the results of gel electrophoresis in the presence of urea and acetic acid2l According to Chalkley at aJL. one phosphate group reduces the mobility of HI by 1.1%. Phosphorlated HI (PH 1) migrated 3-4
Fig. I. Polyacrylamide gel electrophoresis. The urea-acetic aci+1system of was used. Panyim and Chalkley (a) Ascites cell histone HI; (b) phosphorylated ascites cell his-
tone Hl; (c) phosphorylated
7H-labeled lymphocyte HI; (d) 3H-labeled unmodified lymphocyte HI.
a
b
c
d
percent slower than unmodified HI (Fig. 1). More than 95% of the 32P-radioactivity recovered after acid hydrolySiS comigrated with phosphoserine in hij£h voltage electrophoresis (4ata not shown).
Filter binding of HI- and PHI-DNA complexes. Histone and 3H-labeled supercoiled or linear Col El DNA were mixed in a w/w ratio of 0.2 ( _ 42 moles HI/mole DNA) in 0.5 ml samples of binding buffer containing increasing NaCl concentrations. The amount of radioactive DNA retained on nitrocellulose filters is presented as a function of NaCl concentration in Fig. 2. The filter binding of histone complexes with supercoiled Col El DNA is shown in Fig. 2a. In agreement with the observations of Vogel and Singer30, we find that most radioactive DNA 2118
Nucleic Acids Research Fig. 2. Filter binding at
,w
different NaCl
con-
centrat ions.
| amples containing 1.2 u H-labeled superhelical (a) or X0-
(b)
linear
Col
El
DNA
"were made up in 0.5 ml of binding buffer with increas-
\
>
.1
ing
\
25-
\
c
salt
> b
75 4
concentrations.
To
samples was added 0 24Fug HI (*), to another set Was added the same amount of PHI (o). Filter binding was performed as described under Methods. One hundred percent corresponds to 25,500 cpm.
\one set
of
50.
1
!
20
i
ia 60
i
i i. 1.0 180 .
100
220mMNQ a
retained by HI at NaCl concentrations of about 120mM. PHI DNA complexes, however, were retained best at 20mM NaCl. Moreover, HI caused the filter binding of maximally 70% of the DNA at 120mM NaCl while more than 90% of the DNA was retained with PHI in 20mM NaCl. We show below that the fraction of filter bound DNA depends on the histone/'DNA ratio. The filter binding of histone complexes with linear 3H-labeled Col El DNA as a function of NaCl concentration is shown in Fig. 2b. Radioactive DNA was best retained at 20mM NaCl by both HI and PH1, although the fraction of filter bound material was again higher for PH1 DNA complexes. At higher NaCl concentrations, the fraction of radioactive DNA retained with HI reached a plateau and then, at 200mM NaCl and above, decreased. A plateau was not observed when PHI DNA complexes were investigated at salt concentrations of 40mM NaCl and above (Fig. 2b). was
The filter binding curve for PHI DNA complexes is rather similar for supercoiled and linear DNA, but this result is not due to nicking of supercoiled DNA by the PHI-preparation uswd, 2119
Nucleic Acids Research since sedimentation through alkaline sucrose gradients demonstrated that the DNA-strands remained covalently closed after exposure to PHI (data not shown). The fraction of filter bound DNA depends on the histone/DNA ratio. The filter binding of radioactive supercoiled Col El DNA was tested at 20mM NaCl at various ratios of HI or PHI to DNA (Fig. 3). More than 90% of the DNA was trapped on the filters at a ratio of 40-60 moles PHI/mole DNA. At a similar molar HI/DNA ratio only about 20% of the DNA was retained. Similar results were obtained when other DNA species were substituted for Col El DNA in the filter binding experiments (Table 1). These results confirm earlier shown in Fig. 2 and reports that animal virus DNA is similar to bacterial plasmid DNA in its ability to react with histone HI51. Filter binding of histone HI.
DITA does not stick to nitrocellulose filters under the conditions used in this work (Fig. 3). Free histone HI, however, does bind to filters.2934. Fig. 4 shows that a low ionic strength, somewhat less HI bound to the filters than at NaCI concentrations above 2OmM when nearly all of the (5H)-HI or Free
100
o
50-~~~~~~~~~~~~
20
40
100 60 80 moles HI/mole DNA
200
Filter binding at different histone concentrations. Fig. Oamples of I. 8ug 2H-labeled superhelical Col El DNA were placed in 0.5 fil of binding buffer with 20mM NaCl. The indicated amounts of HI (o) were added to one set of samples. A second set received the indicated amounts of PHI (o). Filter binding was performed as described. One hundred percent corresponds to 58,600 cpm. 3.
2220
Nucleic Acids Research Table 1. Filter binding of HI-DNA complexes. The experiments were carried out as described in the text for Col El DNA. Two points should: be noted: 1) the filter binding of polyoma DNA complexes with HI or with PHI is very similar to that of histone Col El DNA complexes§O. 2) under identical conditions, a larger fraction of phage T7 DNA than of sheared mouse DNA is retained on filters. This result probably ref«Rcts the preference of HI for larger over smaller DNA molecules'. ug histone ter/ug DNA
DNA
% bound to filter PHI HI
20
38
80
60
95 62
0.28
50
16
58
0.11 0.28
20
60
85
50
37
53
polyoma DNA
0.35
sheared mouse DNA
phage T7 DNA
(3H)-PHI
NaCl mM
applied to the filters was bound.
Nonradioactive supercoiled Col El DNA was added to 3H-labeled HI and PH1, respectively, before the complexes were passed through nitrocellulose filters. We found a considerable reduvtion in the 3H-labeled histones retained on filters at NaCl concentrations below 20mM (Fig. 4). This result implies that, at low ionic strength, not only HI-DNA complexes29, but also PHI-complexes are formed which do not stick to the filters. Centrifugation analysis of HI-DNA complexes. The fact that more DNA is retained on filters with PH1 than HI at 20mM NaCl (Fig. 2 and 3) could mean that PHI has a stronger affinity for DNA than does unmodified HI. The results of Fig. 2 and 3 could also mean that PHI binds to a larger fraction of the DNA molecules available. The following experiments were performed to test these possibilities.
(3H)-HI and (>H)-PHI
were mixed with unlabeled
supercoiled
Col El DNA at 20mM NaCl and centrifuged through sucrose gradients containing 20mM NaCl. The gradients were collected through a quartz cuvette to identify the position of the DNA by its absorbance at 260 nm (Fig. 5). Radioactive HI was determined in each fraction of the gradient. As shown in Fig. 5b, nearly all of the radioactive HI was 2121
Nucleic Acids Research Fig. 4. Filter binding of
(3H)-Hl.
Upper part: To 0.6 ml samples of binding buffer with the indicated NaCl concentration were added 2.8,ug (3H)-HI. The samples w6re filtered through nitrocellulose filters as describ-
I
l
_.
a ._
1I 1.
,
10
20
30 mM NoC
40
50
ed. The filter bound radioactive HI was registered (e). In a parallel experiment 8 ug unlabeled superhelical Col El DNA was added to the (5H2-Hl containing samples. Filter bound radioactivity was determined (o). Lower part: This experiment was performed exactly as the one de cribed above except that (?H)- Hl was used instead of (5H)-Hl. Filter bound radioactive histone in the absence of DNA (.), filter bound radioactive histone in the presence of unlabeled superhelical Col El DNA (o).
recoverd in those fractions of the gradient which contained the DNA. Similarily, 80% of the PHI was also associated with DNA; the remaining PHI in this experiment was found near to top of the gradient (Fig. 5c). (These experiments have been repeated at different histone/'DNA ratios from 10-100 moles histone,/,mole DNA and at salt concentrations between 2OmM-IOOmM with results essentially the same as those presented in Fig. 5). Thus HI appears to bind at least as well to DNA as PH1 does. In another set of experiments, radioactive supercoiled Col El DNA was mixed with HI or PHI in binding buffer with 20mM NaCl, both at a ratio of 1.05/ug histone/57ug DNA (,v 44 moles histone/mole DNA). The complexes were sedimented through sucrose gradients containing 20mM NaCl. Samples (0.2 ml) were removed from each fraction of the gradient and precipitated with trichloroacetic acid to determine the total radioactivity present. Another 0.2 ml sample of each fraction was diluted 1:5 in filter binding buffer containing 20mM NaCl and passed 2122
Nucleic Acids Research
Fig. 5. Sedimentation
a
ana-
lysis of histone
a2
DNA'complexes in 0.1
1000-
20mM NaCl.
b
.a
E
/
Unlabeled supercoiled Col El DNA (20 ug) in 0.4 ml binding buffer containing 2OmM NaCl (a) without histone, (b) with 4 ug (3H)-HI, and (c) with 4 u; (3H)-PH1 were centrifug4d in sucrose gradients
X
in
the
SW4I
rotor
for 36,000 rpm 4 1/4 hr. CL c ~~~~~~~~~C The relatively high background of ultraviolet light ^ material (smooth absorbing r 60. line) is due to the bovine 02 U serum albumin present in
,_--__-- -__ 5ne-_- _-__ _0 E~~~~~~~~l
% 8
at
_______aS the binding z_
200
dioactivity
slo
15
and 20
buffer. was
The
ra-
determined
in trichloroacetic acid
top nprecipitates bars).
(horizontal
frao
fraction number
through a nitrocellulose filter. The results (Fig. 6) show that, at this NaCl concentration, 32% of the DNA was retained in the presence of HI (Fig. 6a) and 72% in the presence of PHI (Fig. 6b). When 0.2 ml samples from the same fractions were diluted 1:5 into binding buffer containing 100mM NaCl, 60% of the DNA was retained by HI and 58% by PHI (Fig. 6c). This experiment has been repeated at molar ratios of histone to DNA between 20 and 80 with qualitatively similar results (data not shown). In another set of experiments, histone-DNA complexes were formed at different ratios of histone to DNA in 100mM NaCl and centrifuged through sucrose gradients containing 100mM NaCl. For example, when 5,ug 3R-labeled superhelical Col El DNA was mixed with 0.8/ug HI or PHI, centrifuged and tested for filter binding we found that with HI, 48% of the DNA was trapped on the filter and, with PHI 44% of the DNA was retained at 100mM NaCl. Filter binding of the same complexes at 20mM NaCl resulted in retention of 25% of the DNA by HI and 60% by PHI (Fig.7). The data presented in Fig. 5,6 and 7 show t)iat HI and PHI bind about equally well to DNA at 20mM and at 100mM NaCl. Nevertheless, more PHI- than HI-DNA complexes bound to filters 2123
Nucleic Acids Research
a
ll l
6
8
t. 6 0 li' g
_
4
8 1I' .4 ~~~E O .CD zz ° 2l
_ 2.
Fig. 6. Analysis of histone DNA complexes by sucrose gradient and centrifugation filter binding. (a) Complexes of Hi and radioactive suDercoiled Col El DNA were formed at 2OmM NaCl and sedimented through a sucrose gradient containing 2OmM NaCl. The SW41 rotor was used. Samples of 0.2 ml were used for tri-
|b ' \ X~~~I It s : l 2 "' ow*a~ t£, ^., \chloroacetic acid precipitation (.), for filter binding x:27 ^%s\ ff _ at 2OmH NaCl (x) and for ° filter at 100mM b NaCl (Ebinding ), respectively. o (b) Complexes of PHI and laffi 8fi fi x beled supercoiled Col El 0 Xl ~~~~~~~~C DNA were sedimented under . 1 3 identical conditions in a 6- .M1g .6 O parallel tube. Trichloroace._ l.tic acid precipitation (o) and filter binding at 2OmM 7E 4(x) T as well as at 100mM s NaCl , ,a (.A) were performed as 0. I. d above. The recovery of radiE oactive DNA was better than 2 E 42I 0
ft
_
L
.
u
I 510 15 fraction number
top
go~~~~~~~9%.
Free uncomplexed radioactive Col El DNA was centrifuged in a third parallel tube. This DNA traveled the same distance (ten fractions frm the top)as the complexes shown above (data not
shown).
at 20mM NaCl. The simplest explanation of these results is that at 20mM NaCl, PH1 is distributed on many or all (depending on the histone/DNA ratio) of the DNA molecules in the population, while binding of HI is restricted to a smaller fraction of the DNA molecules. Furthermore, unmodified HI appears to bind to a smaller fraction of the DNA molecules at 20mM than at 10imM
XaCI. It should be noted that the experiments of Fig. 6 and 7 are possible because, at the histone/DNA ratio chosen, the sedimentation rate of histone DNA complexes (Fig. 5b and c) did not differ much from that of free DNA (Fig. 5a). (Free DNA was always sedimented as a control in a parallel tube when experi2124
,^;* /1 analyzed as described in the U l a olegend to Fig. 6 except that Z 100mM NaCl was used in the \ i binding buffer and in the su.4.4 |Al crose gradient. After sucroC se gradient centrifugation of the HI-Col El DNA com.2plexes aliquots assayed _wI |q*^ by trichloroacetic acid pre-
o
x
were
,C_..D_*_
20mM NaCl
b
*8-
R CDR
.
.\, t
x 8 6
._ E "
cipitation (e), filter binding at 100mM NaCl (A4) and
c
centrifuged through.
sucrose
gradients containing 100mM NaCl. Aliquots were assayed by
@
(x).
(b) PHI-DNA complexes were formed at 100mM NaCl and
acid
precipitation
(o)
and filter binding at 100mM (A) and 20mM (x) NaCl.. l lThe recovery of radioactive DNA was about 90%. E
4--
2.-~~~~~~~~~~~~a 5
0l 15 fraction number
top
ments of the type shown in Fig. 6 and 7 were performed. At the histone/DNA ratios used in these experiments, no difference in sedimentation rate between free and complexed DNA was observed This analysis, of course, was not sensitive enough to detect possible small differences). This result is surprising since a cooperative binding of HI ot DNA at a w,w ratio of, for example, 0.4 caused filter binding of about 30% of the DNA present (Fig. 3). This fraction should contain DNA molecules complexed with somewhat more than an equal mass of histones. Since this mass increase did not cause an enhanded sedimentation rate we tentatively conclude that the frictional coefficient of the complexes must be higher than that of free DNA, possibly due to a reduction of the degree of superhelicity or to an alteration of the structure of the superhelical DNA (interwound superhelix to toroidal superhelix or vice versa). These complications may be avoided by studying the sedimen2125
Nucleic Acids Research tation of linear Col El DNA-histone complexes. Mixtures of linear 3H-labeled Col El DNA and either or PHI in binding buffer containing 20mM NaCl were centrifuged through sucrose density gradients as described above for the experiments of Fig. 6 and Fig. 7 (Fig. 8). In contrast to the results with supercoiled DNA, the peak fraction of the HI complexes with linear DNA sedimented about 1.3 times faster than free DNA. The PHI-DNA complexes sedimented in a sharp peak about 1.1 times faster than free DNA. On the average 47% of all recovered DNA was retained on nitrocellulose filters by HI and 75% by PH1, respectively. When the filter binding of individual fractions of the gradients was tested, we found that almost 70% of the fastest sedimenting fraction of the HI-DNA complexes was retained but less of the slower sedimenting material bound to filters (Fig. 8a). How-
HI
la
1004
e5
50
c
Fig. 8. Sucrose gradient of histone aanalysis complexes with linear Col El DNA. (a) As a control, linear 3Elabeled Col El DNA was cen-
-*
4
6
A_
.
trifuged through
a sucrose
gradient containing 20mM NaCl
(.).
Complexes of
.2 ug of linear labeled DNA with 0.4 ug of HI were sedimented ih a parallel tube(\). I
.'x
e"
l
l
X !t\Acid precipitable radioactiv2 *
