Volume 14 Number 6 1986
Nucleic Acids Research
Salt induced B - A transition of poly(dG)-po4y(dO and the stabilization of A form by its
methylalion
Yoshifumi Nishimura, Chikako Torigoe and Masamichi Tsuboi
Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Received 3 December 1985; Revised and Accepted 14 February 1986
ABSTRACT
Raman spectra of poly(dG) •poly(dC) have been observed In aqueous solutions
at various Ionic strengths, [NaCl] = 0. 03 to 4 M, and at different temperatures,
10 to 60 °C. At 30 °C, and at [NaCl] - 0.03 M, it was found to have a B-form
(with O^endo-anti guanosine and C2iendo-anti cytidine), whereas, at [NaCl] =
4 M, an A form (with C3,endo-antl guanosine and C^endo-antl cytidine). At 30°C
and [NaCl] = 1 M, namely at an intermediate state, a fraction of this molecules was
considered to have a "heteronomous A" form (with O^iendo-antl guanosine and C3!
endo-anti cytidine). At 60 °C and [NaCl] = 1 M, It assumes the B form, and at
10 °C and [NaCl] = 1 M, the A form. Cytoslne-5-methylation was found to cause a
marked stabilization of the A form. Even at [NaCl] = 0.1 M (at 30 °C), a substantial portion of poly(dG) -poly(dm^C) was found to have a heteronomous form, In
which the dG atrand Is In the B form and the dC an A form; It never assumes a
complete B form.
INTRODUCTION
In contrast to poly[d(G-C)] •polytdfG-C)], there has so far been little knowledge of the conformation of poly (dG) • poly (dC) In Its aqueous solution.
Recently,
we could detect an A form of poly(dG) •poly(dC) In Its concentrated (10% w/w)
aqueous solution by Raman spectroscoplc measurements1.
In continuation of this
work, we have now Investigated In detail the polymorphic forms of poly(dG) • poly
(dC) and poIy(dG) -poly(dm5C) in aqueous solutions at various conditions In the
light of the structure-spectrum correlations which were derived by Raman spectroscoplc examinations of many of the crystals of mononucleotides and nucleosldes ' .
We report here evidence that poly(dG) • poly(dC) undergoes a transition from the
B form to the A form with an Intermediate form by changing the salt concentration
and by changing the temperature of the solution.
We also found that Its methylated
derivative prefers the A form, rather than B form. Our findings lead to a suggestion as for an implications of ollgo(dG) -oligo(dC) tracts which are found frequently
in natural DNAs and also of the methylatlon of DNA In vivo.
© IRL Prest Limited, Oxford, England.
2737
Nucleic Acids Research
EXPERIMENTAL
The sample of poly(dG)-poly(dC), poly[d(G-C)] • poly[d(G-C)], poly(dG)poly(dm 5 C), and poly[d(G-m 5 C)] • poly[d(G-m 5 C)] were all purchased from P . L .
Biochemlcals, Inc.
The sample of poly(dG) • poly(dC) was purified by dialysis of
Its aqueous solution against Na-cacodylate buffer (pH 7.0) and then by lyophlllzatlon.
Its purity was checked by its circular dlchrolsm (CD) spectrum 4 ' 5 .
In 20 mM
NaCl + 2 mM Na-cacodylate buffer (pH 7. 0), It showed a positive band at 255 run,
a crossover at 244 nm, and negative bands at 235 and 213 tun, indicating that no
triple stranded form was Involved.
Another piece of evidence for the absence of
Its triplex formation was obtained by our Raman spectroscoplc examination.
If It
1
were triple helical, Its PO2 symmetric stretching line at 1100 cm" would split Into
a doublet as we found for poly(rU) -poly(rA) -poly(rU).
Our poly(dG) -poly(dC)
sample, however, gave only one sharp line around 1100 cm
condition examined In the present study.
was used without further purification.
in every experimental
The sample of poly[d(G-C)] • poly[d(G-C)]
The sample of poly(dG) • poly(dm 5 C) was
purified by dialysis against Na-cacodylate buffer (pH 7.0) and lyophlllzatlon.
5
The
5
sample of poly[d(G-m C)] • poly[d(G-m C)] was dlalyzed against EDTA-NaOH
aqueous solution (pH 7.0) and then lyophlllzed.
Raman spectroscoplc measurements were made on a Jasco R-800 Raman
spectrophotometer connected with a DECLAB 11/23 minicomputer.
Each DNA solu-
tion, 3% in concentration (w/w), was sealed In a glass capillary tube, and excited
by the 514.5 nm beam of an NEC model GLG3300 Ar ton laser.
Each spectrum was
obtained by accumulation of over 16 scans with 5 data points per 1 cm" 1 .
tral silt width was set at 4 cm~l.
The spec-
After the Raman spectroscoplc measurement, pH
value of each sample solution was examined, and found to remain at about 7.
RESULTS AND INTERPRETATIONS
Figure 1 shows the Raman spectra of three states of poly(dG) • poly(dC) along
with the spectra of the B and Z forms of poly[d(G-C)]-poly[d(G-C)] (Fig. I, d and
e).
The spectrum of poly(dG) -poly(dC) In 0.03 M NaCl solution (Fig. l c ) r e s e m -
bles that of the B form of poly[d(G-C)] •poly[d(G-C)] (Fig. Id).
The low salt
form of poly(dG) •poly(dC) gave characteristic Raman lines at 831 cm" 1 assignable
to a B-type backbone conformation 6 " 8 , at 684, 1317, 1334, and 1365 cm" 1 assignable to an O4iendo-antl guanoalne conformer 2 ' 3 , and at 1242, 1258, and 1272 cm" 1
2738
Nucleic Acids Research
poly(dG)
poly(dC)
AM
NaCI
1M
0.03M
poly(dGdC)
0.1 M
1500 1A00 1300 1200 900 800
Wavenumber( cm"1)
700
600
Fig. 1. Raman spectra of poly(dG) -poly(dC) and poly[d(G-C)] • poly[d(G-C)]
In different ionic strength at 30 °C. ( a ) , Poly(dG) -poly(dC) in 4 M NaCI, 17 mM
Na • cacodylate buffer (pH 7.0). (b), Poly(dG)-poly(dC) in 1 M NaCI, 17mMNacacodylate buffer (pH 7.0). (c), Poly(dG)-poly(dC) In 0. 03 M NaCI, 17 mM Na_cacodylate_buffer^pH 7 . 0 ) . (d), JBoly[d(G-.C)J --polyl^G-C)] in.0.1 MiJaCl
aqueous solution (pH ~ 7 . 0 ) . (e), Poly[d(G-C)] -poly[d(G-C)] In 4 M NaCI
aqueous solution (pH ~ 7 . 0 ) . The capital letters of A, B, and Z In this figure
denote the characteristic Raman lines of A, B, and Z forms. In parentheses, the
assignments of these lines are shown; G, C, and Bk mean the vibrations of guanos Ine,
cytidIne and backbone, respectively. See also Table 1.
2739
Nucleic Acids Research
Tabla 1.
Itolacule
Charactarlatlc Ranan line
• u c l e l c Acids with tha C'C basa pairs.
C+
state*
C
f o m s of
C+
BI +
C
1256
808
C+
Rsferancas
781
790
662
This work
1) A form
"1312
1320
poly(dC)-poly(dC)
B.S.
1388
(1365)
poly(dC)-poly(doe 'O
U.S.
1390
(1353)"1"
1310
(1300)
1320
1257
808
776
662
This work
CpC-Ca
Xtal
1385
(1365)
1321
1293
1248
8L2
784
668
Raf. 7
CpC-Sa
Ital
1388
(1350)
1321
1294
1244
812
784
668
Raf. 9
poly(G)-poly(C)
L.S.
1375
(1365)
1320
1294
1252
815
787
668
Ref. 10
L.S.
1371
(1356)
1316
1300
1258
813
777
669
Raf.
10
L.S.
(D20)
1380
(1360)
1315
1300
1260
810
783
667
Raf.
11
L.S.
1380
(1350)
1320
1296
1254
814
784
669
Raf. 12
1295
(D 2 0)
poly(rC-dC)poly(rC-dC)
r(CCCGCC)2
5'C«P-m(C3'aiido- mtl)
Ital
1394
(1345)
1316
(806)
667
Rafs • 2 , 3
5'dGW-Hl(C3'«ndo-antl)
Ital
1389
(1343)
1311
1321
(805)
664
Rafs • 2 , 3
Cytldlne(C3'endo- mtl)
Xtal
1292
1248
5'CMP-Cd(C3'ando- . « «
Ital
1315
1260
1258
11)
790
Rafs • 2 , 3
(810)
787
Rsfs •
807
835
776
754
684
(661)
This work
1242 831
784
684
This work
830
784
682
This work
1239 836
786
755
682
This work
682
Refs • 2 , 3
673
Refs . 2 , 3
bataronooous A fora
poly(dG)•poly(dme C)
L.S.
1364
1333
1310 (1300)
poly(dC)-poly(dC)
L.S.
1365
1334
1317 'l294
1272
1258
poly[d(G-C))poly[d(C-C)]
L.S.
1366
1334
1319
L272
1259 1239
poly(d(C-«.'c)]pol»[d(C-ae*C)]
L.S.
1366
1334
1316 (1300)
1272
1262
1317
111)
8 fom
5'dGKP-Ha2(04'ando-antl)
Ital.
1364
1333
Guanoslna(Cl'axo-jmtl)
Ital.
1368
1340
5'GMP-Ha2(C2'ando- astl)
Ital.
1365
1327
5'CMP-Zn(cre.xo-ai t l )
Ital.
SHadaoxycytldlna
(C2'ando-antl)
Ital.
5'dQO>-Ba 2 (c3'aio- antl)
Xtal.
1Y)
2 , 3
1293
(879)
(828)
1306
1286
(135O)"1"
1300
1265
1266
(826)
1238
1251
Rafs • 2 , 3
786
Refs • 2 , 3
792
Refs • 2 , 3
(889)
782
Rafs • 2 , 3
1232
1232
1292
677
Z fom
s
poly[d(C-C)Jpoly(d(C-C)J
B.S.
1358
1318
poly[d(C-»i*C))poly(d(C-«a i C)]
U.S.
uJ
1318 (1300)
1293
1266
1246
812
866
7B4
790
624
This work
1266
1246
812
866
780
753
624
This work
784
790
635
Raf. 12
r(CCCCCC)2
B.S.
1350
1318
1290
1266
1245
810
870
d(CGCCCC)2
Xtal.
1358
1320
1284
1268
1246
810
865
785
795
625
(675)
Raf. 13
.<•.'<«*.'«>,
Xtal.
1355*
1310 (1300)
810
(865)
782
750
622
(673)
Raf. 14
2740
(1266) (1246)
Nucleic Acids Research
Tablt 1 . ,
continusd
Holtcule
G+
state*
d*oxyguanosine
(C3'endo-syn)
Xtal.
3'5'cGMP(C4W.yn)
1345
Xtal.
1360
Xtal.
*
+
+
C+
C
BK+
C
C+
Refarenc e a
1318
617
681
Refa. 2 . 3
1318
633
674
Refs.
2, 3
Refs.
2, 3
(767)
1310
1264
1232
(811)
791
The state in which the s p e c t n m is obtained. H.S.; la a high salt solution, Xtal; in a crystalline
etatt, L.S.; in a low aalt eolution.
These Raman lines are characteristic of the local conformations of nucleic acids.
A nethyl deformation vibration of 5—oedeoxycytidine should be located around h«ra.
assignable to a C2tendo-antl cytidine conformer^ (Table 1, cf. lines under the heading (111) B form).
Therefore, it is clear that poly(dG) -poly(dC) takes B form
under low salt condition.
In 4 M NaCl, the spectrum of poly(dG) -poly(dC) (Fig. la) Is markedly different from that at 0.03 M NaCl (Fig. lc ).
The strong Raman line at 808 cm" 1 in
this spectrum is identified with what has been well known as a marker line of A-type
DNA backbone conformation6"8.
The characteristic lines of nucleoslde local con-
formations are observed at 662, 1312, 1320, and 1388 cm" 1 for a C3iendo-anti
guanosine conformer, and at 1256 cm" 1 for a Cgiendo-antl cytidine conformer
(Table 1, top line).
These characteristic lines are all similar to those observed
in the Raman spectra of several nucleic acids whose structures are known to be of
A-type 9 " 12 (summarized In the upper portion of Table 1).
In addition, we found
that the fibrous sample of poly(dG) •poly(dC) at 75% relative humidity, which Is
known to be In the A form 15 , gave similar spectral features (Fig. 2a). It should
be pointed out here that the local conformation of nucleoslde In all of the A form
ollgonucleotldes, for which slnglecrystal X-ray analyses 16 " 1 ^ exist, is found to be
C3iendo-anti.
Also, this Is the case in most of the proposed A-form models 1 9 ' 2 ".
Taking this into account, the high salt form of poly(dG) • poly(dC) can be established
to be the A form.
At 1 M NaCl, an intermediate concentration, both of the characteristic Raman
lines for O4Tendo-antl and C3iendo-anti guanosine moieties are observed, at 684 and
662 cm" 1 respectively (Fig. l b ) . Therefore one might consider that both the A and
B forms are coexisting here.
However, the spectral feature of the 1200-1300 cm" 1
range, which reflects the cytidine local conformation, Is found to be almost the same
as that of the A form (at 4 M NaCl), but not like a mixture of A and B forms. At
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Nucleic Acids Research
poty(dG)
•polyWQ
fiber
(75°/8r.K)
1M NaCl
solution
10° C
30°C
60°C
900 800 700
Wavenumber( cm-1)
Fig. 2. Raman spectra of poly(dG) • poly(dC) In different states and at different temperatures. (a), Fibrous sample of poly(dG) -poly(dC) at 75% relative humidity (a), (b-d), Poly(dG)-poly(dC) In 1 M NaCl, 17 mM Na• cacodylate buffer (pH
7.0 ) at 10 °C (b), at 30 °C (c), and at 60 °C (d ).
[NaCl] = 1 M, therefore, the poly(dC) moiety of poly(dG) -poly(dC) la considered
to be In the A form, whereas the poly(dG) moiety Is in the B form In half of the
duplex molecules In the solution.
In other words, In the course of the Ionic strength
change (low to high) the poly(dC) strand precedes the poly(dG) strand in the B -* A
transition, and an intermediate form In this B -* A transition may have a heterono
mous structure, in which dG and dC strands adopt B- and A-type conformations
respectively.
Such a structure was actually found in the crystal of d(GGCCGGCC ) 2 2 1 .
The terminal GG sequence of the octamer in this crystal at -8 °C has a B-type con-
2742
Nucleic Acids Research
formation; the torsion angles around C41-C31 bonds (or 3 ) of both guanosine moleties are about 120°.
This 3 value corresponds to an O^iendo sugar puckering rather
than a C3iendo ( 3~90°) or a C2,endo ( 3 ~150°) puckering.
In contrast, the CC
sequence, the base-pairing partner, has an A-type conformation with <5 values of 90°
corresponding to C3rendo puckering: the GG/CC duplexes in the both ends of the
octamer duplex in the crystal at -8 °C take a heteronomous structure, and the Intermediate form of the B -* A transition of poly(dG) •poly(dC) discussed above may be
similar to this structure.
We also Investigated the temperature effect on this B -» A transition of poly
(dG) -poly(dC) (see Fig. 2).
It was found that poly(dG) -poly(dC) In 1 M NaCl
aqueous solution at 60 °C takes the B form (Fig. 2d ) and on lowering the temperature
to 10 °C It transforms into an A form (Fig. 2b), through the intermediate form which
was partly detected in the spectrum at 30 °C (Fig. 2c ).
This temperature depend-
ence of the conformation is analogous to that of d(GGCCGGCC>2 In Its crystalline
state 21 .
At -8 °C, its terminal dlnucleotlde duplex portions with the GG/CC
sequence in the crystal adopts a heteronomous structure, as stated above, while at
a lower temperature, -18 °C, the whole duplex assumes a complete A form
.
5
Figure 3 shows the Raman spectra of three states of poly(dG) -poly(dm C)
along with the spectra of the B and Z forms of poly[d(G-m5C)] •poly[d(G-m5C)]
(Fig. 3, d and e).
Although the vibrations of 5-methyldeoxycytldlne in methylated
DNA around 1350 and 645 cm"1 gave some different spectral profiles from those of
unmethylated DNA, the other portions of the spectra d and e of Fig. 3 are quite similar to the spectra d and e of Fig. 1, respectively.
5
Therefore, the B and Z forms
5
of poly[d(G-m C)] -poly[d(G-m C)] are considered to have almost the same local
conformations as the B and Z forms of poly[d(G-C)] • poly[d(G-C)], respectively
(lowest four lines of Table 2).
Our structure-spectrum correlations (indicated in
Table 1) are considered to be applicable also to the methylated DNA. Thus, the
characteristic Raman lines of cytldlne conformation are assumed to remain unchanged on the methylatlon.
At 4 M NaCl, poly(dG) • poly(dm5C) gave the characteristic Raman lines for
A-type strutifilre as shown In Fig. 3a and Table 1.
Thus, poly(dG) • poTyfdm^C)
is also found to take A form at high salt condition.
However, at 0.1 M NaCl condi-
tion, It gave a different spectrum (Fig. 3c) from that of the B form of poly[d(G-m5
C)] (Fig. 3d ).
A characteristic Raman line for the cytldlne conformation Is still
2743
Nucleic Acids Research
poly(dG)
• poly(dmC)
AM
NaCl
1M
0.1M
poly(dGdmC)
0.1 M
1M
1500 1400 1300 1200 900 1 800
Wavenumber (c rrr )
600
Fig. 3. Raman spectra of poly(dG)-poly(dm 5 C) and poly[d(G-m 5 C)]-poly[d
(G-m C)] Indifferent Ionic strength at 30 °C. ( a ) , Poly(dG) • poly(dm 5 C) at 4 M
NaCl, 17 mM Na• cacodylate buffer (pH 7 . 0 ) . (b), Poly(dG)-poly(dm 5 C) at 1 M
NaCl, 17 mM Na• cacodylate buffer (pH 7 . 0 ) . ( c ) , Poly(dG)-poly(dm 5 C; at 0.1
M NaCl, 17 mM Na-cacodylate buffer (pH 7.0). (d), Poly[d(G-m°C)] • poly[d(Gm 5 C)J at 0.1 M NaCl aqueous solution (pH 7.0). (e), Poly[d(G-m 5 C)] • poly[d(Gm 5 C)] at 1 M NaCl aqueous solution (pH 7. 0). Raman lines assignable to the methyl
group vibrations of 5-methyldeoxycytldtne are Indicated by *.
5
2744
Nucleic Acids Research
Table 2.
Polymorphic forsi of DNA with C-C baae-palr* as rerealed
by our Eaaun ipectroscoplc study.
DHA
Backbone
coofomat loo
Condition
dC
Poly(dC)-poly(dC)
Structure
Sugar p uckering and
glycoayl coafonmatlon
dC
Low s a l t
B
04 '•ndo-antl
C2 'endo-anti
lotcrTNdlat*
salt
A+B
04 'endo-anti
C3'endo—anti
High sale
A
C3 '•ndo-anti
C3'«ndo-anti
A
C3'endo-anti
htttronoaous
A
C3'endo-anti
A
C2'endo-anti
B.*
9
Poly (dG)« poly (dM C)
Poly[d(G-C)]-poly(d<G-C)]
Poly[d(G-»e 5 C)]'poly[d(G-»e 5C)}
Low s a l t
A+B
M 'endo-anti
<C3 'endo—iotl)
High salt
A
C3 '•ndo-anti
1
Low s a l t
B
04 endo—antl
High salt
Z
C3 'endo—iyn
Low aalt
8
04 'endo-anti
Intcraadlat*
•alt
1
+ Thli «Cructure l i an A fom with different
~
C3 'enao-sya
Bh*
heteronomous
A+
C2'endo-Cl'exo-anti
Z
B.1
C2'endo-anti
C2'eodo-Cl'•xo-anti
Z
iugar puck«n b«tw««n doubl* itrandi at obierved In th« temlnal
dlnucUotlde duplai of d[CCCCGGCCl2 crr«tal at -8*C 2 1 .
T Thlf ttructure l i i 1 form vlth alternating augar puckara.
observed at 1258 cm" 1 indicating that a C^iendo-antl cytldlne remains In this polymer even at 0.1 M NaCl concentration.
The spectral feature of the 600-700 cm" 1
1
and 1300-1400 cm" ranges reveals that an O^rendo-anti guanos In e conformer
coexists as a main component with a C3iendo-antl conformer as a minor component.
Two marker lines are clearly observed at 807 and 835 cm" 1 for A- and B-type backbone conformations respectively.
It is concluded that the methylated poly(dG) • poly
(dC) cannot take a complete B form even at low salt condition and its low-salt form
is a heteronomous structure mainly with the dG and dC strands of B- and A-type
conformations respectively.
This may be similar to the intermediate form of the
unmethylated derivative, described above.
DISCUSSIONS
Our present findings are summarized in Table 2.
In contrast to poly[d(G-C)] •
poly[d(G-C)] which shows the salt Induced B — Z transition, poly(dG) -poly(dC)
shows a salt-induced B -» A transition,
m the formerB-* 2 transttion, no apparent
intermediate form was detected, while in the latter B -* A transition, an Intermediate
form in which the two strands have different conformations was found.
C-methyl-
atlon of poly[d(G-C)]-poly[d(G-C)] stabilizes the Z form, while the C-methyl2745
Nucleic Acids Research
atlon of poly(dG) •poly(dC) stabilizes Its A form.
These findings are Interesting In
comparison with the results of crystallographic analyses which show that the DNA
ollgomers with CG sequences are crystallized Into Z forms 2 2 ' 2 3 , while ollgomers
with GG/CC sequences are crystallized Into A form.
16
17
d(CCGG)2 , d(GGTATACC)2 ,
d(GGCCGGCC)221.
The examples of the latter are
d(GGGGCCCC)218
and the modified A form of
This parallelism suggests that, in general, the crystalline
state and the high salt aqueous solution are similar environments for the nucleotlde
molecules ; In both of them the activity of water Is lower.
Our findings make it possible to understand some properties of poly(dG)- poly
(dC) In solutions under various conditions.
It was once found that X-ray scattering
Intensity versus scattering angle curves of 5% poly(dG) • poly(dC) In solution could
be well explained by adopting the radius of gyration of A form rather than B form 24 .
Although the precise salt concentration used In that experiment Is not clear, It Is
probably that what the author observed was the "A form" or the "heteronomous A
form" which Is found In our present Raman experiment. By the use of the bandshift
method25> in their electrophoresls experiment, Peck and Wang determined the helical repeat of ollgo(dG) -ollgo(dC) tracts Inserted In the plasmld to be 10.7.
This
Is an Intermediate value between 10 (B-form) and 11 (A-form), and Is understandable If the structure of the ollgomer portion is assumed to have the "heteronomous
A form", proposed In our present study.
DNase n digestion experiment Indicated
that ollgo(dG) -ollgo(dC) duplex has asymmetric conformation In which the dG strand
Is much more easily attacked by the enzyme than the dC strand
.
This conforma-
tion may also be the "heteronomous A form" we propose In which an O^endo-antl
guanoslne and C3iendo-antl cytldine are Involved.
Under conditions of lowered
water activity, however, both strands are known to be equally sensitive to DNase II
digestion.
This is understandable because here the ollgo(dG) •ollgo(dC) duplex Is
considered to take the A form as shown In our work, and both strands should have
the same local conformations.
In our study, it has also been demonstrated that the methylatlon of cytostne
causes the GG/CC duplex portion of DNA to prefer the A form.
The methylatlon of
DNA In vivo has been considered to play a role In the regulation of the gene expression2''.
The methylatlon Is frequently found In the CG sequence, and the stabiliza-
tion of the Z form In the methylated DNA has been considered to have a biological
2746
Nucleic Acids Research
Importance^.
slte^?.
However, CCGG sequence is also found to be an in viyo methylation
d(CCGG)2 duplex was crystallized as an A form
and the methylation of
GG/CC sequence causes to favour A form in aqueous solutions as shown in our study.
The B ->• A transition of the methylated DNA may therefore also be related to some
biological function.
ACKNOWLEDGEMENTS
This work was supported by a grant (No. 57060004) from Ministry of Education, Science, and Culture of Japan.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
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