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Cooperative B Z transition of' poly(dG tiC?) induced by ,spcz,'mlnc'" at physiological temperature in aqueous medium M , V , R , I ( a o . M. A t r e y i a n d Shashi S~lxena I ) ¢ l ) a r t m r n t o f ('h('mi,~t£v. [ "nivcr~;t v o f Ih-Illi, I)).llti - I I 0 O0 ?. I m l l .

I(cccivcd 13 ,luly lqqO: revised version rccci~cd 30 ()ctobcr Iq~}t) Sl'leflil|ll¢ illdtl¢Cd I~ /, Irdll'illlOli o r pol}ld(i d ( ' ) in ~,tqn¢otl~ 111~2~.1i0,11|1;l[ a l l d zl[~)/,." lah}siolol:'.ic.'d [Clllperd|Ht'¢.'. ill cclltlli~,r COIl~¢¢ltlf;,llJOl} levels I11 lOW i o n i c ~.[r¢l~glh ill¢(,|lt|nl T h e 11111i11¢to p h o s p h a t e t'~lto. A P, a! the Ii'lld[)oll'~t oi" t h e t f;lil'~JliOl'l dcct'c~lscs ~,~ilh III¢I'C;I'sC II1 t e t l l p e r a t t l t c , "I'hc CllIh,tlpy ¢ll;tll/'~c ~.s'~ 45 L,"; kc;tl l)cr I)tOl of foopt'f;4tl~C tlflll, the [l'.tl~lf*ltlOl) it~dtl¢cd I~,' sl~¢l'lllll|¢2 IS hJl~hlv ¢t)opc'r;|l, ivc ',~ith t| coopcrall~'¢ tint[ leltlHh nf 7or) 7:20 ,d '~ll'(' ('tl'ctll.|t'diClll'oJsH1; Polyld( ' ,If 'l: I] Z [f;tllsJliofl; [!lhJetllLIn)bl'Ollfide

1 INTRODUCTION T h e t r a n s f o r m a t i o n of p o l y ( d G - d C ) to left handed f o r m can readily be induced by choosing a right combination o f solution parameters [1-5]. Some chemical modifications of bases also facilitate the transition [6]. In this context, we earlier reported that the biogenic tetraanaine, s p e r m m e , effects B - Z transition o f pol y ( d G - d C ) , at levels as tow as those f o a n d in cells, m 5O'/o e t h a n o l / d i o x a n e [71. We now report that slight reduction in water activity is not necessary for a highly c o o p e r a t i v e B - Z transition if the t e m p e r a t u r e is raised to physiological t e m p e r a t u r e . 2. M A T E R I A L S A N D M E T H O D S Spermine (4HC1) was fronl Sigma and p o l y ( d G - d C ) was from Pharmaci~l (Lot No. PD717910). Solutions o f the polynncleotide ( - 1 0 - s M phosphate) were prepared in pH 7.0 S$C buffer. 3 m M in NaCI, 0.3 m M in s o d i u m citrate. Circular dichroic (CD) spectra were recorded with a J A S C O J-500A unit,

3. R E S U L T S Titrations of p o l y ( d G - d C ) with spermine were carried out at four different temperatures, 20, 30, 37 and 50°C, and CD spectra were recorded as a function of a d d e d polyamine. At 20°C, the tetraamine caused aggregation of the D N A above an amine to D N A p h o s p h a t e ( A / P ) ratio o f 0.9, and below this ratio, no p e r t u r b a t i o n of the C D spectrum was observed. At 37°C, the CD s p e c t r u m remained unchanged till an A / P o f 0.1, but thereafter, the intensity o f the long Correspondence address: M . V , R , Rod, D e p a r t m e n t o f C h e m i s t r y , University o f Delhi, Delhi-110 007, India

Published by Elsevier Science Publishers B. V. {Biomedical Divisioto

wavelength band as well as that o f the 250 nm negative b a n d sharply decreased, and the bands were ultimately replaced by a negative band at 293 nm and a positive band at 265 nm, which are characteristic [2] o f tcft handed Z-form o f p o l y ( d G - d C ) (Fig. 1). A plot o f the cllipticity at 293 nm as a function of A / P shows (Fig, 2) that the transition is quite cooperative, and the midpoint of the transition is at an A / P o f 0.17. The amount of spermine required for shifting the B - Z ectuilibrium to the right decreased as the temperature was raised to 50°C (Fig. 2) and the midpoint o f the cooperative transition was at an A / P of 0.11. When the titration was carried out at 30°C, there was only a partial reversal o f the positive 280 mn band and the D N A aggregated above an A l P o f 0.76. 3.1. Enthalpy qf transition and cooperative unit length The isodichroic point at 273 n m in the C D spectral titrations seen both at 37°C (Fig. 1) and also at 50°C, indicates clearly that the transition involves two species. The state o f eauilibrium between the two conf o r m a t i o n s , at a gwen addition o f the tetraamine, but at different temperatures could conveniently be comp a r e d f r o m the value o f K = oe/(l -ce) where ce is the fraction o f D N A in Z - c o n f o r m a t i o n [8,9]. The C D spectra o f a system containing 2.5 × 10 -5 M o f p o l y ( d G - d C ) and 3.9 × 10 -6 M o f spermine ( A / P = 0.181) were recorded in the temperature range 2 0 - 8 0 ° C . The plot of InK versus 1/T (Fig. 3) was linear, and the enthalpy change was calculated to be 45 ± 8 keal per tool of cooperative unit; thezaG a n d A S values at 37°C are respectively, - 0 . 7 5 and 0.149 kcal per tool o f cooperative unit, The cooperativity length u0 was established following the procedure of F r a n k - K a m e n e t s k i i , Ivanov and

63

.2 4

I._.

230

. ,190

..-_-L.---I_._L_L_J 270

290

310

X(nm!

coworkers [lo-121, from the data c-111the cffcct of the tie, ethidium bromide, on the midpoint and width of the transition, using the expression

where As is the initial width of the transition curve, &7, and 6Aa are respectively chc shangc in the transition midpoint and width of the transition curve, in the presence of the tie and C is the number of mol of the tic per mol of base pairs. Titrations of poly(dG-dC) with spermine were carried out at 50°C in the presence of the tie (C = O.OOl-0.005 mol of tie per mol of base pairs). The cooperativity length was calculated to be 700 * 20 base pairs. 4. BEXXJSSION The present data show that spermine at physioiogisal temperature is a very effective inducer of the Zconformation for poly(dG-de). Further, of the 3

-25

Fig,

64

v 005

wp

02s

ocla

2. E!lipticity at 293 nm versus A/P at 37°C and 50°C.

cellular ~~0lyi~lllill~S, 0llly thd t~trilillllifl~ IlilS d Iii& sp0zificity to stabilize the %.-form. In fact, the di- and fri-amincs, putrcscinr and spcrmitlinc, RI’C incffectivr in inducing the transition CvrII ilt 5O*C; Wtually, while sprrniidine Ci\LlSCS an aggregation to 9” form (A/P > 6-S), putrcscine only precipitates the DNA at very high A/P ratios (r22). The high specificity of spcrminc to stnbilisc the Zform of poly(dG-dC) is associntcd with a low Jar-1value an.d ii large cooperative unit Icngth of 700 bp, Such n large cooperative unit Iength was observed for poly(dA-dC) - poly(dG--dU) only when both the pyrimidine bases were modified to mSC and 13rsU, and that too in high salt (4.37 M) Incdium but dH was also large (480 kcal). For chemically unmodified poly(dG-dC), .ql for the isocnthalpic change in 2,2§ M salt was reported to be 300. The cooperative unit length is even smaller (1 10) for IV@’ (1 mM) induced transition at high temperature (76.6”C, dH: 307 kcal) [13]. And YO was only 25 for the ethanol-indused transition [5]. However, the l3-2 transition of chemically utlmodified d(CpG),, inserts in plasmids, brought about by negative supercoiling, is reported to be highly cooperative [ 14,151, III many of the studies on alternating purine-pyrimidine systems, the right to left handed transformation is achieved only under forced coaditions like high salt, low water activity or chemical rnodifisation of the pyrirnidine bases. On the other hand, our results demonstrate that the transition oscurs under physiologisal conditions of temperature and concentration of the ceilular tetraamins spermine. It is also of interest to note that the Ieft handed form yields at high A/P an aggregate rk” form, characterized by an intense positive CD band at 250 nm. The above transitions are reversed by enhancing the ionic strength of the medium as iwdicated below, 37”C;A/P:O.17 w

A/P:0.90 4Z------+PF+-Z-B

90 mM

150 mhl

NaCl

NaCl

VohllliC 278, i1Ltf~lbef I

FEllS I.E'I"I'IH~,S

It i;: signit'icanl t h a i n o l OIdy Ihc COlll'oi'maliOll h u l the stale o[ purinc-pyrimidinc

al;grcgation o1' polynuclcotM¢,

lhc al(crimtin~ at p h y s i o l o g i c a l

tcnlpcralttr¢, is a rtln¢(ioi~ of bolh ionic slrci~glh, al~d ¢o1~¢cnlralion of spcrminc; lhc variation in the Icvcls of spcn~dnc with cell ,~,,rowth is wcll documclucd [16l,

J a l l u a r y 1991

I?1 IL~o, M . V . I t , Alrc)i, M. ~md .'-iax~n,h S, {1990) I|iopol)'m~x 'hJ I.lq~ big?, K,II. told ,~la¢~It¢, MA:,

I,'1I lhdl *I

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Ileal l.a~uHdn, Ymg., l:railk-l(anlcncl~l~ii, M.I). and Tdfonov. [!.N. 11970) lliopolym~r~ 9, 1253.-1.I(~i. V.I.. Mindlcnkova. Io.I!.. MinyaL £.I!.. Frank. K'ullcllct~kii, M,I), alld Sihyolkilul, A,K, 1197.1) J. M~I. Iliol. ~7, ~ 17--I153, Nliny;~t. E.F'... Iv'-m(~v. V.I.. Krilzyu. A,M,, Minchcnk~va, I..E. and Schyolkin:h A.K. (1971~)J. Mol. Biol. 12B. 397..o.109. Soull pasi~, I),M mid J,avit~. T.M. 11987) Nucleic Acid~ and

1111 h;m~v, REFERENC[.:S {I] Pohl. I:.M. and Jo~in. T..M. (H)72).I. M{~I. Biol. 67. 375~J06, {2] llch¢. M. :rod Fchcrffcld. (i. (l,~.Sl) PI'~c, Nail, Acad. S