volume 9 Number 211981
Nucleic Acids Research
The B-»Z transition of poly(dG-dC).poly(dG-dC) modified by some platinum derivatives
Bernard Malfoy, Brighte Hartmann and Marc Leng
Centre de Biophysique Moleculaire, C.N.R.S., 1A, avenue de la Recherche Scientifique, 45045
Orleans Cedex, France
Received 25 September 1981
SUMMARY
Poly(dG-dC).poly(dG-dC) was modified by chlorodiethylenetriamino platinum
(II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammine
platinum (II), respectively. The conformation of these modified poly(dG-dC).
poly(dG-dC) was studied by circular dichroism. In 4 M Na + , the circular dichroism spectra of poly(dG-dC)<iiew-Pt (0 < rj, « 0.2) are similar (rb is the
amount of bound platinum per base). It is concluded that the conformation of
these polymers belongs to the Z-family. Dien-Pt complexes stabilize the Zform. The midpoint of the Z ->• B transition of poly(dG-dC)dien-Pt(0.12) is at
0.2 M NaCl. Moreover another B •* Z transition is observed at lower salt concentration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stability
of Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of small
amounts of EDTA. Poly(dG-dC).poly(dG-dC) modified by ais-Pt and trans-Pt complexes do not adopt the Z-form even in high salt concentration.
The therapeutic efficiency of cis-dichlorodiammine platinum (II) (eis-Pt)
on tumor is now well-established. It has been demonstrated that cis-Pt binds
to DNA and several lines of evidence suggest that this binding is related to
anti-tumor activity of this compound (general reviews, 1,2 and references
herein). Numerous studies have been carried out in an attempt to describe the
modification of DNA. In vitro, ais-Pt compound binds strongly to DNA and guanine residues are the most preferred binding site (1,3,4). The recent discovery that oligo(dG-dC) crystals (5-7) and poly(dG-dC).poly(dG-dC) fibers (8) can
adopt the Z-form lead us to ask the question whether the binding of cis-Pt to
poly(dG-dC) .poly(dG-dC) could hinder or favour the B •+ Z transition. It has
been already shown that the covalent binding of some products to guanine residues in poly(dG-dC).poly(dG-dC) favours the Z-form (9-12). Moreover, as already done by other investigators, it seemed to us of interest to compare the effects of two other platinum compounds, trans-dichlorodiammine platinum (II)
(trans-Pt) and chlorodiethylenetriamino platinum (II) chloride (dien-Pt) which
have no anti-tumor activity.
In this paper, we report some results obtained by circular dichroism on
poly(dG-dC).poly(dG-dC) modified by these three platinum compounds. It is
© IRL Press Umited. 1 Falconberg Court. London W 1 V SFG. U.K.
Nucleic Acids Research
known that in solution poly(dG-dC).poly(dG-dC) can undergo a reversible salt
induced conformation change with a midpoint at about 0.7 MgCl_ or 2.5 M NaCl
(13). The low-salt form was shown to belong to the B-family (14) and the highsalt form to the Z-family (13,15-18). Circular dichroism is very convenient to
study these two forms. The circular dichroism spectrum of the high-salt form
is almost an inversion of the spectrum of the low-salt form (13,19). In this
work, as judged by circular dichroism, we show that cis-Pt and trans-Pt complexes prevent the transition of poly(dG-dC).poly(dG-dC) to the Z-form. On the
other hand, we found that poly(dG-dC).poly(dG-dC) modified by dien-Vt compound
undergoes two transitions from the B-form to the Z-form or Z-like form and
that these two transitions occur at much lower salt concentrations than that
of unmodified poly(dG-dC) .poly(dG-dC) .
MATERIAL AND METHODS
Poly(dG-dC).poly(dG-dC) bought from P.L. Biochemicals was treated with
phenol and then exhaustively dialyzed as already described (11). Platinum compounds were a gift of Dr. J.P. Macquet (Toulouse). The quantitative fixation
of platinum compounds on poly(dG-dC).poly(dG-dC) was performed as described by
Macquet and Butour (20). The platinum compounds were dissolved just before
used in 10 mM NaClO^ and added to a 200 ug/ml solution of poly(dG-dC).poly(dGdC) in the same medium. The reaction was run at 37°C in the dark for 24 hours.
The modified samples were exhaustively dialyzed against 1 mM phosphate buffer,
pH 7.3. The platinum content of some samples was determined with an atomic
absorption spectrometer (21). We will call r, the number of platinum atoms
bound per nucleotide. We will write poly(dG-dC)cis-Pt(0.15), poly(dG-dC)transPt(0.15) and poly(dG-dC)diew-Pt(O.15) a sample of poly(dG-dC).poly(dG-dC) complexed respectively with eis-Pt, trans-Pt and dien-Vt at r b of 0.15.
Methylation of guanine residues of poly(dG-dC).poly(dG-dC) was performed
as already described (22). Dimethylsulfate (Aldrich) (total volume 6 yl) was
added in three times every 30 minutes to a solution of poly(dG-dC).poly(dG-dC)
(0.5 mg in 1 ml of 0.5 M sodium cacodylate, pH 7). The product was then exhaustively dialyzed against 1 mM phosphate buffer. The percentage of modified bases was determined by gel filtration chromatography on Sephadex G-10 after
acid hydrolysis of the polymer (23) .
Ultraviolet absorption (UV) and circular dichroism (CD) spectra were recorded with a Cary 210 spectrophotometer and a Roussel Jouan III dichrograph,
respectively.
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RESULTS
The formula of the three compounds which were respectively reacted with
poly(dG-dC).poly(dG-dC) are the following :
H2C_CH2
Cl
H3N
\
Cl
HN
NH 2
\
NH 3
H2N
Cl
H3N
\
Ptt
/
Pt
/
Cl
\
H3N
Cl
cis-Pt(NH 3 ) 2 CI 2
[pt(dien)Cl]ci
trans-Pt(NH 3 ) 2 CI 2
Dien-Pt compound has only one reactive function while cis-Pt and tvansPt have two.
1) Dien-Pt compound
In figure 1, we compare the CD spectra of poly(dG-dC).poly(dG-dC) and of
poly(dG-dC)<f£en-Pt at various r,, in 1 mM phosphate buffer.
The CD spectrum of poly(dG-dC).poly(dG-dC) presents a positive band and
A£
At
2 ^290
8
4
--2
-<
/
0.05 0.1 0.15 rb
2
/
£?"%•
6
\
\
2
<\
-2
\
/ / ?.
•••••'
//
\
-4
A
v. i
•+'•••"+/
-2
. —-'
+ +/
•
/
-
V
>-'
250
300
WAVELENGTH
-6
B
250
300
nm
Fig. 1 - Circular dichroism spectra. Poly(dG-dC)dien-Pt at various r D . Medium
rb = 0 ; —
r b = 0.05 ; ••• r 0 = 0.08 ;
1 mM phosphate buffer pH 7.3. A)
rjj = 0.12 ; +++ r^ = 0.22. Inset : variation of Ae290nm a s a function of
r b . B) +++ r b = 0.22,
rb =0.25 ;
r b = 0.30.
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Nucleic Acids Research
then a negative one as already reported in the literature (19). The binding
of dien-Pt compound to poly(dG-dC).poly(dG-dC) modified greatly the CD spectrum. As rb increases, the first positive band decreases and then becomes negative while the negative band becomes positive. At rj, = 0.12 the spectrum of
poly(dG-dC)dien-Pt(0.12) is almost an inversion of the spectrum of poly(dGdC).poly(dG-dC) . The shape of the spectra is almost the same for 0.12 < r^ <
0.2. At larger values of r,, new changes were observed in the spectra. At r^=
0.3, the spectrum presents an intense positive band centered at 295 nm and
then a negative band (figure IB).
As shown in the inset of figure 1A, the largest absolute value of Ae.gg
was found at r. = 0.12. This modified sample was studied in more detail.
The CD spectra of poly (dG-dC)cKerc-Pt(O.12) recorded in various media are
shown in figure 2. In 4 M NaCl (or NaClO^) the CD spectra of poly(dG-dC)dt:enPt(0. 12) and of poly(dG-dC).poly(dG-dC) are identical (for sake of clarity,
the spectrum of poly(dG-dC).poly(dG-dC) is not shown). In 1 mM phosphate buffer, the spectrum of poly(dG-dC)<2ien-Pt(0. 12) is slightly different from that
in 4 M NaCl. The first negative bands are identical but the positive band of
-4
-6
250
300
WAVELENGTH nm
Fig. 2 - Circular dichroism spectra of poly(dG-dC)dien-Pt(0.12) as a function
of salt concentration. NaCl concentration :
0.1 mM ; ••• 5 mM ;
30
mM ; +++ 100 mM ; —•— 1 M and 4 M. All the solutions contain 1 mM phosphate
as a
buffer pH 7.3. Inset : variation of ^290
function of the logarithm of
sodium concentration.
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the spectrum in 1 mM phosphate buffer is less intense and red-shifted. In 30
mM NaCl the spectrum is completely different, the first band being positive
and the second one negative.
The transition between these different forms are cooperative. This is
shown in the inset of figure 2 where the variation of A£2go (other wavelengths can be used) is plotted as a function of NaCl concentration. The midpoint of the first transition is at about 6 mM NaCl and of the second transition at 0.2 M NaCl.
Addition of EDTA destabilizes this low-salt form of poly(dG-dC)dien-Pt
(0.12). The CD spectrum of poly(dG-dC)die«-Pt(0.12) in 1 mM phosphate buffer
plus 0.1 mM EDTA is identical to that in 30 mM NaCl (positive band and then
negative band).
2) Cis-Pt compound
The CD spectra of two poly(dG-dC)cis-Pt, in 1 mM phosphate buffer, are
shown in figure 3A. The spectrum of poly(dG-dC)eis-Pt(O.15) presents a small
negative band at 295 nm, then a positive band centered at 275 nm and then a
positive band at 225 nm. Even at higher r, , there was no inversion of the CD
spectrum. All the experiments were done within two days after the modification.
Some evolutions in the CD spectra were observed as a function of time. After a
few days, the first negative band became smaller and the first positive band
A£ . A
4
.
B
2
-2
-i.
250
300
WAVELENGTH
250
nm
300
Fig. 3 - Circular dichroism spectra. A) poly(dG-dC)eie-Pt, B) poly(dG-dC)iransPt ;
rb = 0 ;
r D = 0.05 ; —•— rj, - 0.15. Medium 1 mM phosphate buffer, pH 7.3.
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Nucleic Acids Research
became larger ( r e s u l t s not shown).
3) Trans-Pt compound
The CD spectrum of poly(dG-dC)ir>ans-Pt(0.15) differs from the spectrum
of poly(dG-dC).poly(dG-dC) but the changes are not so drastic as those found
with dien-Ft and eis-Pt complexes (figure 3B). The first positive band is redshifted and the intensity of the negative band is smaller with a small redshift of the minimum. Changes in the spectra were also observed as a function
of time.
Finally we have studied poly (dG-dC).poly(dG-dC) modified by the three
platinum compounds respectively in 4 M NaCIO,. The spectra of poly(dG-dC)dienPt(0.12), poly(dG-dC)cis-Pt(0.15) and poly(dG-dC)ferans-Pt(0.15) are shown in
figure 4. The spectra of poly(dG-dC).poly(dG-dC) and poly(dG-dC)dien-Pt(0.12)
are identical and very different from those of poly(dG-dC)eis-Pt(0.15) and of
poly(dG-dC)fcrons-Pt(0. 15) . Similar results were obtained in 4 M NaCl.
4) Methylated poly(dG-dC).poly(dG-dC) and poly(dG-dC).poly(dG-dC) in ethanol
In order to explain some of our results (see discussion) we have studied
on one hand a methylated poly(dG-dC).poly(dG-dC) and on the other hand poly
(dG-dC).poly(dG-dC) in presence of ethanol.
Poly (dG-dC).poly(dG-dC) was reacted with dimethylsulfate and about 30 %
of the guanine residues (on the N(7)) were modified. The CD spectra in 1 mM
At
3
»*
N
1
•
s
7
•
*
*
*
\
•
\
*
\
-1
-3
250
300
WAVELENGTH nm
Fig. 4 - Circular dichroism spectra. •• • poly(dG-dC)dien-Pt(0.12),
poly
poly(dG-dC)cis-Pt(0.15). Medium 4 M NaClO^ plus
(dG-dC)trans-Pt(0.15 ;
1 mM phosphate buffer, pH 7.3.
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phosphate buffer and 4 M NaCl are shown in figure 5. The spectrum in 4 M NaCl
looks like the spectrum of poly(dG-dC).poly(dG-dC) in the same solvent. The
low salt form •*• high salt form transition is cooperative and the midpoint is
about at 2 M NaCl (inset, figure 5 ) .
The stability of poly(dG-dC).poly(dG-dC) in presence of ethanol was also
studied by CD. It has been already shown that addition of alcohol induces the
B ->• Z transition (19). In 1 mM phosphate buffer, 0.1 mM EDTA results similar
to those already published (19) were found. The midpoint of the transition is
at 60 % ethanol. The experiment was repeated in the same buffer without EDTA.
The midpoint of the transition is at about 35 % ethanol (results not shown).
DISCUSSION
In this work we compared the conformation of poly(dG-dC).poly(dG-dC) modified respectively by the two bifunctional compounds cis-Pt and trans-Pt and
by the monofunctional compound dien-Pt. These compounds bind strongly to poly
(dG-dC).poly(dG-dC) but their effects are very different.
It has been found that guanosine-5'-monophosphate and cytidine 5'-monophosphate coordinate to dien-Pt through N(7) and N(3) respectively (24). In
At 290
/
4
'
\
'.
i
1
"1
-3
-5
= I
-3
2
-2
-1
0
lo,[Na*]
/
-2
-4
W
S
/
250
300
WAVELENGTH, nm
Fig. 5 - Circular dichroism spectra. Methylated poly(dG-dC).poly(dG-dC) (0.3).
1 mM phosphate buffer pH 7.3 ;
4 M NaCl plus 1 mM phosphate buffer
pH 7.3. Inset : variation of Ae290nm a s a f u n c t i ° n °f t n e logarithm of sodium
ti
concentration.
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Nucleic Acids Research
double stranded helices, the N(3) of cytosine residues are not accessible and
thus are not expected to react. This has been verified on poly(I).poly(C) (25,
26). In the reaction between nucleic acids and cis or irons-platinum compounds
the N(7) of guanine residues are probably involved (1,2). However, bidendate
complexes can be formed and it is not yet known with certainty whether one or
two nucleotide residues react.
Let us discuss first the results obtained on poly(dG-dC).poly(dG-dC) modified by dien-Pt compound.
In 4 M NaCl (or NaClO^) the shape of the CD spectra of poly(dG-dC)dien-Pt
(0 ^ r, < 0.20) are similar. These spectra have a first negative band centered
at 292 nm and then a positive band centered at 262 nm. They are almost an inversion of the CD spectrum of poly(dG-dC).poly(dG-dC) in 1 mM phosphate buffer.
Several results show that the high salt conformation of poly(dG-dC).poly(dGdC) belongs to the Z-family (13,15-18) and thus we conclude that poly(dG-dC)
dierz-Pt (0 < rfe < 0.20) in 4 M Na adopts the Z-form.
Dien-Pt complexes stabilize the left-handed helix. The midpoint of the
B •+ Z transition for poly(dG-dC)dierc-Pt(0.12) is at about 0.2 M NaCl (2.5 M
NaCl for poly(dG-dC).poly(dG-dC) (13)). We can only speculate on the mechanism
by which dien-Pt complexes increase the relative stability of the left-handed
helix. One can assume an alteration in dipole moment and polarizability due
to the bound dien-Pt, a charge effect (modified guanine residues bear two positive charges), a steric hindrance in the B form, the existence of hydrogen
bonds between the NH- or NH groups of the dien-Pt and the adjacent nucleotides
or between a phosphate group and the hydrated dien-Pt in the Z-form and not
in the B-form. All these factors can play a role.
The dten-Pt residues are bulky and,as already noted (5), the N(7) of guanine residues are more accessible in Z-form than in B-form. On the other hand,
the conformation of DNA is hardly altered by the binding of dien-Pt residues
as judged by CD (20,27).
It has been already reported that dien-Pt complexes with poly(I).poly(C)
are thermally more stable than poly(I).poly(C). It was proposed (26) that hydrogen bonds are formed between the amine groups of the dien-Pt residues
and the adjacent bases. Hydrogen bonds might be more efficient in Z-form than
in B-form of poly(dG-dC)dien-Pt.
Modifications of guanine residues by mitomycin (9) or by acetylaminofluorene residues (10-12) stabilize the Z-form. The covalent binding of acetylaminofluorene residues to the C(8) of guanine residues favours the syn conformation of the modified nucleotides (28,29) which can stabilize the Z-form
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(in the Z-fonn, the guanine residues have the syn conformation (5)). It is not
yet known whether the binding of dien-Pt to the N(7) of guanine residues can
favour the syn conformation.
To look at the charge effect we compared poly(dG-dC).poly(dG-dC) and a
poly(dG-dC).poly(dG-dC) sample in which about 30 % of the bases were methylated (on the N(7)). There was no change in the CD spectrum of the methylated
sample in the range 1 mM-1.5 M salt. An inversion of the CD spectrum was observed at higher ionic strength. The midpoint of the transition is at 2 M NaCl
(2.5 M NaCl for poly(dG-dC).poly(dG-dC)). The methylated sample and poly(dGdC)dien-Pt(0.12) have about the same number of positive charges. However, each
modified guanine residue bears two positive charges in poly(dG-dC)<£ien-Pt as
compared to one in the methylated poly(dG-dC).poly(dG-dC). This might explain
the larger effect of dien-Vt.
The CD spectra of poly(dG-dC)dten-Pt(O.12) and of poly(dG-dC).poly(dG-dC)
in high salt concentration are very similar and we concluded that the two polynucleotides have the same conformation. This conclusion has been confirmed by
immunochemical studies (30 and work to be published). In 0.1 M NaCl, 1 mM
MgCl2 the antiserum of a rabbit immunized with poly(dG-dC)dien-Pt(0.12) electrostatically bound to methylated bovine serum albumin, precipitates poly(dGdC)d£en-Pt(0.12) and does not precipitate poly(dG-dC).poly(dG-dC). At high
salt concentration, poly(dG-dC).poly(dG-dC) and poly(dG-dC)dien-Pt(0.12) are
precipitated by the antiserum.
The midpoint of Z + B transition of poly(dG-dC)<i£e?3-Pt(0.12) is at about
0.2 M NaCl. As the NaCl concentration was still decreased, there was a new
cooperative transition (midpoint at 6 mM NaCl). The CD spectrum of poly(dG-dC)
dien-Pt(0.12) in 1 mM phosphate buffer looks like that in 4 M NaCl. However,
the positive band is less intense and red-shifted (figure 2 ) . Because of the
similarity between the spectra in low (1 mM) and high (4 M) ionic strength,
we assume that in 1 mM phosphate buffer we are still dealing with a lefthanded helix, the geometry of this helix being slightly different from that in
4 M NaCl (or NaC10 4 ).
The stability of this low-salt form is decreased by addition of EDTA.
Wang et at. (31) have described a Zj.-form in oligo(dC-dG) crystals. In this
Z .-form, some phosphate groups form hydrogen bonds to hydrated magnesium ions
complexed to the N. of guanine residues. It is tempting to speculate that in
the low-salt form of poly(dG-dC)dten-Pt(0.12), the dien-Yt residues bound to
the N7 of guanine residues form a bridge through water to close phosphodiester
oxygens. This would be a major stabilizing factor which could be destroyed by
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EDTA and NaCl.
It is not possible to verify by immunochemical experiments that in 1 mM
phosphate buffer a Z-form is observed. In this low ionic strength, non specific interactions occur between the antiserum and double stranded polynucleotides.
The B •* Z transition of poly(dG-dC) .poly(dG-dC) is also affected by the
presence of small amounts of EDTA. The midpoint of the B •* Z transition induced by addition of ethanol is at about 60 % in presence of 0.1 mM EDTA (in
agreement with the literature (19)) and at 35 % in absence of EDTA. We have
previously reported that in 1 mM phosphate buffer, the percentage of Z-form in
poly(dG-dC).poly(dG-dC) modified by acetylaminofluorene residues is dependent
upon the presence of EDTA (11). These results can be understood assuming that
small amounts of multivalent ions can stabilize a Z-form (traces of multivalent ions are always present despite the use of twice distillated water and
first grade reagents). This is also in agreement with the results of Behe and
Felsenfeld (32) who found that some multivalent ions are very efficient to
induce the Z-form in poly(dG-m dC).poly(dG-m dC).
As more dien-Vt complexes are bound to poly(dG-dC).poly(dG-dC) (r, > 0.2),
a new transition occurs. The CD spectrum of poly(dG-dC)dien-Pt(0.3) has an intense positive band centered at 295 nm and then a large negative band. Work is
in progress to elucidate this new conformation.
Cis and trans
platinum complexes do not seem to induce the Z-form. The
analysis of the results is difficult because there are some changes in the
spectra as a function of time (it should be noted that no changes were observed with poly(dG-dC)dien-Pt). In 1 mM phosphate buffer, poly(dG-dC).poly(dGdC) modified by cis and trans platinum compounds are not in the Z-form. Moreover, even in 4 M Na + , poly(dG-dC)c?:s-Pt(0. 15) and poly(dG-dC)trans-Pt(O.15)
do not adopt the Z-form. Cis and trans platinum complexes prevent the B •+ Z
transition of poly(dG-dC).poly(dG-dC) probably by inter or intrastrands crosslinks.
ACKNOWLEDGEMENTS
We thank Professor C. Helene for his interest in this work. The comments
of Dr. W. Guschlbauer, Dr. J. Ramstein and Dr. E. Sage are appreciated. We are
indebted to Dr. J.P. Macquet (Toulouse) for the gift of platinum compounds.
This work was supported in part by Delegation Ggngrale 3 la Recherche Scientifique et Technique, contract n° 79-7-0064.
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