Download Conformation of DNA in chromatin protein

volume 6 Number 4 April 1979
Nucleic A c i d s Research
Conformation of DNA in chromatin protein-DNA complexes studied by infrared spectroscopy
J.Uquier, M.C.Gadenne, E.Taillandier*
Laboratoire de Spectroscopie Biomole'culaire, U.E.R. de Medecine, University Paris XIII, 74, rue
Marcel Cachin, 93000 Bobigny, and
N.Defer, F.Favatier, J.Kruh
Institut de Pathologie Moleculaire, 24, rue du Fg Saint-Jacques, 75014 Paris, France
Received 19 December 1978
ABSTRACT
The following observations concerning the DNA secondary structures in
various nucleohistone complexes were made by infrared spectroscopy : 1 / in
chromatin, chromatin extracted by 0.6 M NaCl, nucleosomes and histone-DNA
reconstituted complexes, the DNA remains in a B type conformation at low rel a t i v e hygrometry ; 2/ in chromatin extracted by tRNA and in non hi stone
protein-DNA reconstituted complexes, the DNA can adopt an A type conformat i o n . Infrared linear dichroTsm data show that in NHP-DNA complexes the low
relative hygrometry conformation of DNA may be modified and that the i n f r a red parameter 8 1 0 9 0 is close to that measured f o r RNA's or DNA-RNA hybrids.
I t is concluded
that the histones block the DNA in a B form and that some
of the NHP could be involved in the control of the secondary structure of
DNA in chromatin.
INTRODUCTION
Chromatin proteins include histones and an heterogenous group of-proteins, the non histone proteins (NHP) ; in rat liver nuclei both types of
proteins are present in approximately equal amount. Double stranded DNA
exists in two main families of conformations : the A and B type helix geometries (1). In aqueous buffers or in fibers exposed to high relative humidities and containing a small amount of trapped salt, free DNA adopts a B type
secondary structure. In vitro under less polar conditions or at low relative
humidities the DNA adopts an A type conformation. The biological signification of these forms as well as the importance of the B •• A transition are
not well understood ; however it would be surprising if this transition had
no implications in vivo. Infrared spectroscopy allows one to identify the
different conformations of DNA using the characteristic absorption bands of
the phosphodiester chain (800 -- 900 cm" region) and the dichroic ratios of
various absorptions in the case of polarized spectra (infrared linear dichroism) (2).
We have previously shown that DNA bound to histones H2A, H2B, H3 or H4
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remains, for a certain histone/DNA ratio blocked in a B type conformation while for the DNA bound to hi stone HI or H5 the B -• A transition occurs with decreasing relative humidity (3,4). It has recently been shown that at least
part of NHP is associated with the chromatin subunits, the nucleosomes (5).
Some NHP are able to bind to DNA and to modify the thermal stability of double stranded DNA (6). This observation has been correlated with the current
assumption that NHP could be involved in the control of gene expression(7,8).
It is then interesting to investigate the possible influence of the binding
of these proteins on the conformation of free DNA and of DNA included in
chromatin.
In this work we show that in NHP-DNA complexes the DNA can undergo the
B -> A transition. However the observed dichroTc ratios suggest that the A type conformation angular parameters of the low relative humidity form are modified. We establish that the DNA in native rat liver chromatin and in nucleosomes remains in a B type conformation at low relative numidity. The effect, on the secondary structure of DNA in chromatin, of the removal of certain NHP or of certain histones by salt and tRNA treatment has also been investigated.
MATERIAL AND METHODS
Association of NHP with DNA. DNA was prepared according to Marmur (9). For
the preparation of the NHP, rat liver nuclei were prepared according to
Chauveau et al. (10), washed 3 times with 10 mM Tris HC1 pH 7.1 CaCl 2 3.3mM
and NHP extracted as described by Kamiyama and Wang (11). NHP-DNA association
was performed as previously described (6), except that the final mixture was
dialyzed against 5mM NaCl instead of 2mM.
Preparation of nucleosomes. Nucleosomes were prepared after digestion of purified rat liver nuclei with staphylococcal nuclease (Worthington) according
to Shaw et al. (12) as described previously (5).
Preparation of chromatin. Liver chromatin was prepared according to the method described by Tata and Barker (13) modified as follows : purified nuclei
prepared according to Chauveau et al. (10) were washed twice with 10 mM Tris
HC1 pH 7.1,CaCl2 3.3 mM, and once with the digestion buffer : 10 mM Tris HC1
pH 8.0, 100 mM NaCl, 1 mM dithiotreitol, 8% glycerol and 0.25 mM CaCl 2 : nuclei were then suspended in the same medium (100 ^ g ^ ^ m l ) and incubated for
90 sec at 37° with 0.2 units of staphylococcal nuclease. The digestion was
stopped by addition of 2 mM EGTA. The suspension was centrifuged at 1000 g
for 5 min., and then at 150 000 g for 2 hrs. For protein extraction with NaCl,
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the chromatin solution was brought to a f i n a l 0.6 M NaCl concentration by addition of 1 mM NaCl with gentle s t i r r i n g . For protein extraction with tRNA,
0.1 M Mg was added to chromatin to a f i n a l 1 mM concentration. A 15 f o l d
excess (with respect to the DNA amount in chromatin) of tRNA dissolved in
10 mM Tris-HCl buffer, pH 7.3 was added with gentle s t i r r i n g to the viscous
solution of chromatin (containing 1.7 mg DNA/ml) according to the procedure
of Bokhon'ko et a l . (14), I l y i n et a l . (15).
Chromatin was then centrifuged at 150 000 g for 2 hours. Chromatin pellets were solubilized in 5 mM NaCl and dialyzed overnight against this same
solution. Proteins were extracted with 5 M urea, 2 H NaCl and analyzed by
electrophoresis performed according to Laemmli (16) on SDS-14% polyacrylamide gels. The gels were stained with Coomassie B r i l l i a n t Blue.
Infrared spectroscopy. The infrared spectra were recorded on a Perkin Elmer
180 double beam ratio recorder spectrophotometer (between 4000 and 700 cm" )
equipped with a wire grid polarizer (KRS 5 Support) placed in the common beam
and oriented at 45° with respect to the s l i t s and the preferential s t r e t ching direction of the samples. The complexes were deposited on I r t r a n 2
windows and when possible, oriented by mechanical unidirectional stroking.
The relative humidities of the I.R. cells were monitored as described previously (3) and the water content of the samples checked by the measurement
of the water absorption band at 3400 cm" . The protein-DNA ratios were cont r o l l e d by infrared spectroscopy using the maximum of the Amide I or of the
Amide I I absorption of the protein and the 1710 cm absorption of the DNA.
The NHP/DNA input weight ratios were f o r the reconstituted complexes 0.2/1
0.4/1
0.6/1 0.8/1 1/1 and 1.5/1. In a l l samples the NaCl/DNA weight
ratio was such that in similar conditions DNA undergoes a B •+ A t r a n s i t i o n .
RESULTS
1 . Determination of the secondary structure of bound DNA by the use of the
800 - 900 cm conformation sensitive Infrared absorptions. The phosphate backbone of free DNA presents in the 800 - 900 cm region absorptions
characteristics of its conformations. The infrared spectra of B family type
DNA present a specific band at 835 cm" . With decreasing relative humidity
this absorption disappears and new bands at 860 and 807 cm are observed
corresponding to the A type geometry of the DNA.To identifie the A conformation of DNA we use the 860 cm" absorption band as its intensity in I.R.
spectroscopy is more important than that of the 807 cm band ; besides, the
baseline of the 860 cm" absorption is less affected by the strong broad wa1481
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ter band around 600 cm . Thus the two conformation sensitive absorptions at
835 cm"* and 860 cm"* are very useful to obtain informations on the secondary structure of DNA even of unoriented samples and we have used them to characterize the conformation of DNA bound to different chromosomal proteins
and in chromatin.
At high relative humidity the infrared spectrum of chromatin presents
in this region a strong band at 835 cm" similar to that existing in free
DNA (fig. la) in the same conditions. Infrared spectroscopy confirms thus
that DNA in chromatin at high relative humidity is in a B type conformation.
However at low relative humidity (fig. lb) this absorption in chromatin remains present although shifted to 830 cm" whereas in free DNA it disappears completely. Thus DNA in chromatin adopts a B type conformation. We must
notice that a weak absorption is nevertheless detectable around 855 cm at
low relative humidity in chromatin supporting the possibility of the existen-
LogB- DNA
A
CHROMATIN
A
CHROMATIN .6M NaCI
NUCLEOSOMES
hor hsr-DNA .6-1
/\
NHP-DNA 1-1
CHROMATIN-
9
h
900
A-DNA
800crri1
Figure 1 : Infrared absorption spectra of nucleoproteins in
the 800 cm"1 - 900 cm"1 region. Samples were exposed to low
relative humidity (66% R.H.) (except for B-DNA : 931 R.H.)
Tot. h i s t , means preassembled core histones.
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ce of a small amount of A type conformation of the DNA but the B •* A structural transition is almost completely inhibited. In a similar manner in nucleosomes (fig. Id) the DNA is in a B conformation at high relative humidity
and remains blocked in this geometry at low relative humidity. However the
characteristic band is shifted from 835 cm to 825 cm" . This displacement
exceedes the experimental error and may reflect a slight modification of the
phosphodiester chain geometry.
In order to investigate the respective role of the chromosomal proteins
in the stabilization of the B conformation we have studied chromatin depleted from different proteins by 0.6 M NaCl and reconstituted NHP-DNA and histone-DNA complexes. The electrophoretic patterns of the proteins extracted
by urea-NaCl treatment of chromatin and of chromatin previously treated with
either 0.6 M NaCl or tRNA are shown in figure 2. We observe that chromatin
exposed to 0.6 M NaCl reveals a loss of histone HI and of certain non histone
proteins, whereas tRNA treated chromatin is depleted from several classes
of non histone proteins. In both case at least 90t of the core histones (H2A,
H2B, H3 and H4) remain present.
The infrared spectrum of 0.6 M NaCl treated chromatin in the 800 - 900
cm" region (fig. lc) is, at low relative humidity, very similar to that of
native chromatin (strong band at 830 cm ). However we observe at 855 cm
an absorption', small, but slightly more important than in chromatin. On the
contrary tRNA treated chromatin presents in the same conditions a totally
different absorption spectrum (fig. lg), similar to that of A type DNA "(fig.
lh) : strong band at 860 cm" , a band at 807 cm" , no absorption at 835 cm .
Thus the B -*• A conformational transition is prevented in 0.6 M NaCl treated
chromatin and becomes possible in tRNA treated chromatin.
Using the same conformation sensitive bands, we have previously observed that the DNA in reconstituted complexes with the histones of the core
particle (H2A, H2B, H3 and H4 preassociated) is at high R.H. in a B type
form. For a histone/DNA input ratio of 0.6/1 (and higher) the DNA remains
blocked in this conformation when the relative humidity is decreased (fig.
le) (J.L., E.T. Manuscript in preparation). It is thus unambiguously determined, that for a histone/DNA ratio even smaller than that existing in native chromatin, the histones stabilize the DNA in a B type conformation. On
the contrary, NHP-DNA complexes, as can be observed in fig. If present at
low relative humidity a typical A type pattern for the DNA. An example of
this B -»• A structural transition is shown figure 3 in the case of a 1/1 NHPDNA complex (this input ratio is close to the NHP-DNA ratio in rat liver
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HI
I
(3)
• H2A, H2B
- H3
- H4
(b)
(c)
Figure 2 : Electrophoresis of chromatin proteins from
rat liver. Chromatin proteins were extracted by 5M
urea 2 M NaCl, (a) from chromatin, (b) from chromatin
previously treated with 0.6 M NaCl, (c) from chromatin
previously treated with tRNA. Electrophoresis were
performed on SDS. 14% polyacrylamide gels according to
Laemmli (16).
chromatin). At high relative humidity (left) the DNA adopts a B type geometry ; with relative humidity (left to right) the B •+ A transition is observed : at intermediate relative humidity (90* to 76* R.H.) both absorptions
at 835 cm'1 and 860 cm"1 coexist ; at low relative humidity (66% R.H.) the
absorption at 835 cm" characteristic of the B form is no more detected
(right). We can therefore conclude that in the NHP-DNA complexes the non
histone proteins bound to the DNA do not prevent the B -*• A conformational
transition.
2 . Further characterization by infrared linear dichroTsm. In order to ob-
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RH
low
Infrared absorption spectra in the 800 an - 900 cm region of
Figure 3
relative humidity decreases from l e f t to r i g h t .
NHP-DNA 1/1 complex
tain additionnal data on the effect of chromatin protein on the conformation
of DNA we have used polarized infrared data to compute dichroTc ratios and
angular parameters for oriented samples.
a/ NHP-DNA reconstituted complexes. The NHP-DNA complexes present a flow
birefringence and can be oriented into dichroTc films. Figure 4 presents the
polarized spectrum of a 0.8/1 NHP-DNA complex. At high relative humidity
(932 R.H.), the main absorptions correspond to the DNA and are observed at
the same frequencies as for free DNA. Super-imposed on the DNA spectrum we
detect the contribution of the proteins at 1625 cm (shoulder at 1650 cm" )
for the Amide I band and at 1545 cm" (shoulder at 1520 cm" ) for the Amide
I I non dichroTc band. One can observe the following features characteristic
of a B type conformation of the DNA :
- a strongly perpendicular band in the 1710 cm"
caused by the AT and GC base pairing.
region considered to be
- a non dichroTc band at about 1230 cm" and a strongly perpendicular band
at about 1090 cm involving the stretching vibrations of the P0,, groups.
At low relative humidity (66% R.H.) figure 5 shows the polarized spectra of NHP-DNA complexes with 0 . 2 / 1 , 0.8/1 and 1.5/1 weight input ratios.
The persistence of the absorption band at 1710 cm" indicates that in our
conditions the DNA in the NHP-DNA complexes is not disorganized at low relative humidity. The following differences with spectra obtained at high relative humidity and concerning the polarizations of the main absorptions are
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RH93>
1600
800 cm
1200
Figure 4 : Infrared polarized transmission spectrum of NHP-DNA 0.8/1 complex
at high relative humidity (93* R.H.). Arrows indicate characteristic DNA and
protein absorptions. From l e f t to right : 1710 cm"1 (DNA), 1625 cm"1 (Amide
I ) , 1545 cm"1 (Amide I I ) 1230 cm" 1 , 1090 cm" 1 , 830 cm"1 (DNA). — Electric
vector of polarized l i g h t parallel to the orientation axis.
Electric
vector of polarized l i g h t perpendicular to the orientation axis.
.2/1
Figure 5 : Infrared polarized transmission spectra of NHP-DNA
complexes at low relative humidity (66* R.H.). Same notations
as figure 4. Top to bottom NHP/DNA input ratios : 0.2/1
0.8/1
1.5/1.
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clearly observed :
- the band at 1230 cm is now perpendicularly polarized instead of non dichroTc.
- the band at 1090 cm is polarized parallely instead of perpendicularly.
These features are characteristic of a spectrum of A type DNA and confirm
the existence of a conformational transition of the DNA in NHP-DNA complexes
already proved by the study of the 800 - 900 cm region.
It has been previously shown that the best I.R. parameter to characterize the A and B conformations is the angle between the transition moment of
the 1090 cm absorption band and the preferential orientation axis of the
sample (3). Table 1 summarizes the values of Q-ingn concerning various NHPDNA complexes. 9 i n Q n is related to the dichroTc ratio R of the band by :
2
^ ^ ^
where g is a parameter related to the orientation factor of the sample. The
dichroTc ratio of the 1710 cm absorption band, for which the orientation
of the transition moment is known, gives the value of the g parameter. The
dichroTc ratios measured on the spectra are corrected for water contributions as well as for protein contributions for high protein-DNA input ratios.
We have considered a model in which the preferential orientation axis of the
sample remains the axis of the DNA double helix.
At high relative humidity Q I Q Q Q remains around 68° for all NHP-DNA input ratios, as in free rat liver DNA in B form, within the limit of experimental error. However at low relative humidity we observe a progressive decrease of Q I Q Q Q when amounts of NHP increase in the samples. Figure 6 shows
the plot corresponding to these low relative humidity configurations. Above
Table 1 : Orientation of the transition moment of the 1090 cm-1 mode with
respect to the DNA axis for different NHP-DNA input ratios.
NHP
Low relative humidity
form (degree)
High relative humidity
form (degree)
0
0.2
51
50
67,5
0.4
0.8
48
45
67,5
1.0
1.5
44
68
68
-jjfj^ weight input ratio
45,5
68
67
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9
60 1090
50
"
LOW RH
\
o
° o
40
mNHP
m'DNA
0.5
1.5
Figure 6 : Orientation of the transition moment of the 1090 cm- i mode of
NHP-DNA complexes at low relative humidity for various NHP/DNA input ratios.
0.8/1 weight input ratio, 6i O gn is stabilized around 44°. The low relative
hygrometry value is therefore clearly modified with respect to that in free
DNA in A form examined under identical conditions.
It is now well established that the mode at 1090 cm" is dominated by
the in phase dioxy 0 ~ P — 0 stretch and the study of the conformational
transition of the DNA shows that the dichroic ratio of this absorption is
sensitive to the different geometries of the DNA brought by rotations
around single bonds and particularly to the orientation of the P0 2 group.
Then, it may be reasonable to correlate the decrease of Q I Q Q Q to a slight
modification of the A type conformation of DNA.
We have verified that this modification at low relative humidity is
due to the presence of the non histone proteins bound to the DNA. A complex
of DNA with NHP treated by RNase (input ratio NHP/DNA : 0.8/1) has issued
the same angular parameter as the corresponding complex prepared with untreated NHP. The decrease of the angular parametere 1O g O is thus not due to
a possible presence of RNA. Another complex was prepared with NHP treated
by pronase : the resulting Siggo was tnat of tne initial free DNA. It is
thus clear that the modification of the DNA geometry in NHP-DNA complexes
at low relative humidity is due to the NHP bound to the DNA.
b/ Chromatin. Although it is not possible to obtain dichroTc films of
liver chromatin and of mononucleosomes, chromatin treated with 0.6 M NaCl
or tRNA presents a certain amount of orientation and becomes dichrofc. In
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all the spectra, the infrared absorptions corresponding to the DNA are located at the same wavenumbers as in free DNA. The protein bands are found
at 1652 cm (very weak shoulder at 1630 cm" ) and at 1545 cm" (very weak
shoulder at 1520 cm ). The relative intensities of these absorptions are
different from those observed in reconstituted NHP-DNA complexes. The relationship of this infrared data with the protein conformations in nucleohistone complexes shall be discussed elsewhere. Figure 7 presents the spectra
of tRNA treated chromatin (a,b) and of 0.6 M NaCl treated chromatin (c,d)
at low and high relative humidities. Table 2 summarizes the variations of
the observed polarizations as well as the values of the computed dichroTc
ratios.
In the case of 0.6 M NaCl treated chromatin the absorptions at 1710 cm ,
1230 cm" and 1090 cm" are respectively perpendicularly polarized, non dichroTc and perpendicularly polarized at high relative humidity. At low relative humidity they become respectively perpendicular, slightly perpendicular
1600
1200
800
Figure 7 : Infrared polarized transmission spectra of : (a)
tRNA treated chromatin (low R.H.). (b) tRNA treated chromatin (high R.H.).
(c) 0.6 M NaCl treated chromatin (low R.H.)
(d) 0.6 M NaCl treated chromatin (high R.H.). Arrows indicate 1
characteristic
DNA absorptions. From left to right : 1710 cm" ,
1230 cm*1 and 1090 cnrl. Same notations as figure 4.
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Table 2 : Polarizations and dichroTc ratios of the 1230 cm" and 1090 cm
absorptions in treated chromatins.
0 : non dichroTc.
// : parallely
polarized.
J_ : perpendicularly polarized.
DNA
.6M NaCl-chromatin
tRNA - chromatir
v cm"
1230
1090
1230
1090
1230
1090
Low
pol
_L
II
_1_
_L
II
R.H.
R
1.15
0.90
weak
J_
1.08
1.17
1.14
0.83
High
pol
0
J_
0
_1_
0
J_
R.H.
R
1.0
1.23
1.0
1.17
1.0
1.20
and perpendicular. Infrared spectroscopy shows that DNA in chromatin depleted from HI and certain NHP by salt treatment acquires oriented parts which
have always the characteristics of a B type DNA helix. Electron microscopy
observations have shown the presence in histone HI depleted chromatin of
beads connected by linear filaments. Thus the slight dichroTsm observed here
could be due to the expansion of the chromatin structure. On the contrary
chromatin depleted to a higher extent from NHP by tRNA treatment presents
oriented parts with various DNA conformations depending on the relative humidity. At high relative humidity (fig. 7b) one observes a characteristic B
form while at low relative humidity (fig. 7a) the A form spectrum is clearly
visible. Since the complete set of histones remains bound to DNA (fig. 2 ) ,
proteins selectively extracted by tRNA appear then to be involved in the
stabilization of the B type DNA helix in chromatin.
DISCUSSION
The infrared spectra have shown that the DNA in chromatin exists mostly
in a B type helix geometry. Furthermore we show that the B -*• A transition is
inhibited in chromatin in opposition to what occurs in free DNA which under
the influence of various external factors is able to change its configuration. The amount of A type conformation increases only very slightly when
chromatin is treated by 0.6 M NaCl or in mononucleosomes. In NaCl treated
chromatin HI and a certain amount of NHP considered usually as beeing responsible for the superstructure of chromatin have been extracted (17). We
can therefore conclude that the A or B type of DNA helix geometry is not
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related to the superstructure of chromatin. The B form has been also recently observed by Raman spectroscopy in chromatin gels and nucleosomes in good
agreement with the present data at high relative humidity (18) (19). The I.R.
spectroscopy has also given an opportunity to examine the role played by the
inner histones and by non-histone proteins in maintening the secondary structure of DNA in chromatin.
He have established in earlier studies that when only one of the histones H2A, H2B, H3 or H4 is bound to DNA, the latter cannot undergo the B •* A
transition any more (3) (4). We have reported in this work that when the
four histones of the core particles interact together with the DNA, the nucleic acid of the complexes remains always in a B type conformation even for
a histone-DNA ratio, smaller than in chromatin. All these results suggest
that the four histones, which form the structural repeat unit, stabilize the
DNA in a B form and are involved in the loss of its conformational flexibility in nucleosomes, chromatin, as well as in reconstituted complexes.
The DNA conformational transitions in chromatin are affected not only
by histones but also by NHP. Moreover there is evidence to support two different influences of the NHP on the DNA conformation and it seems possible
to correlate this observation with the existence of two subgroups of NHP as
far as the control of the B + A transition is regarded. In our study it is
clear that certain non histone proteins are able to bind directly to the DNA
but leave possible the B + A transition. We shall call this fraction (NHP) A .
Moreover we can notice that the value found for the Qjngg parameter in these
(NHP)A-DNA complexes (44°) is comparable to those of double helical RNA's
(40°±4°), synthetic polyribonucleotides (42°±2°) or triple helical poly(A+2U)
(46°±2°) (20) (21).
In a similar manner the DNA in tRNA treated chromatin is in an A type
conformation at low relative humidity although DNA structure in native chromatin is always of B type (tRNA treatment as shown in figure 2 leads to the
removal of a part of the non histone proteins while leaving all histones
bound to the DNA). Under our experimental conditions in samples containing
a large amount of histones the dissociation of an NHP fraction which may be
weakly bound to the DNA allows the double helix to undergoe the B •+ A transition (reconstituted histone-DNA complexes in similar conditions remain
blocked in a B type conformation). These results suggest that some NHP remOved by tRNA treatment, which we shall call (NHP) , are, in the presence of
histones, responsible for the inhibition of the B •* A transition. The remon
val of these (NHP) from chromatin also increases its solubility and the DNA
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accessibility to RNA polymerase (14). We find that the (NHP) are necessary
for the chromatin DMA to remain blocked in a B form. It is thus possible
P
that the (NHP) are preferentially bound to the histones and stimulate the
hi stone ability to block the ONA in a B conformation. Another possibility
is that the function of the (NHP) may be to overcome the histone influence
on the DNA structure and that this function is inhibited by the presence of
P
the (NHP) . In conclusion, the non histone proteins play an active role on
the secondary structure of DNA. We have shown that the NHP which are directly bound to the DNA slightly modifie the helix geometry of the A family
conformation. Moreover, when histones are present, the NHP control the B -»• A
transition of DNA. It has been suggested that the B •+• A transition is involved in the transcription processes. It could be assumed that the chromatin
proteins which influence this transition could play a role in the control of
gene expression.
AKNOWLEDGMENTS. This research was supported by grants from the CNRS (ATP
chromatin) and from the INSERM.
*To whom all correspondence should be addressed.
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