Interaction of Z form poly(dG-dC). poly(dG-dC) with divalent metal ions: localization of the binding sites by I.R. spectroscopy

Volume 12 Number 10 1984 Nucleic Acids Research Interaction of Z form poly(dG-dC).poly(dG-dC) with divalent metal ions: localization of the binding sites by I.R. spectroscopy J.A.Taboury, P.Bourtayre, J.Liquier and E.Taillandier* Laboratoire de Spectroscopie Biomoleculaire, UER de Medecine, Universite Paris XIII, 74, rue Marcel Cachin, 93000 Bobigny, France Received 5 April 1984; Accepted 1 May 1984 The secondary structures of poly(dG-dC).poly(dG-dC) in the pre- + sence of2alcaline, alcaline earth and first row transition metal ions (Na , Mg2+, Co + , Ni2+) are investigated by infrared spectroscopy. The conformational transitions are studied as a function of the hydration of the polynucleotide and counter-ion nature and content. The use of selectively deuterated poly(dG-dC).poly(dG-dC) on the 8-carbon of guanines allows to show that a direct interaction occurs between the N7 site of guanines and the transition metal ions Co 2 + and Ni 2 + . In the case of Mg 2 + , for high ion/nucleotide ratios, the interaction occurs essentially at the level of the phosphate groups. This interaction leads to a modification of the left-handed conformation. Based on the IR spectroscopy results, an explanation is proposed for the different efficiencies of these various ions to induce the B -+ Z transition. INTRODUCTION In synthetic polynucleotides, the existence of left-handed helixes, first suggested by U.V. circular dichroism experiments (1), has Deen confirmed by X ray crystal diffraction studies (2,3) and characterized by other various techniques : P and H NMR (4-7), Raman (8,9) and IR spectroscopy (9,10). In native systems, the existence of such conformations has been evidenced by indirect immunological techniques (12-14). This novel conformation 1s now extensively studied, because of its possible biological importance : hypothesis have been proposed concerning the role played by Z DNA in fundamental processes such as regulation of gene expression (15). Among the various parameters involved in the B -»• Z transition of poly(dG-dC). poly(dG-dC) in solution, the nature and the concentration of various ions has been previously studied by U.V. absorbance and circular dichroism (1,16-19). On the other hand, the important role played in vivo and in vitro by metal Ions in processes Involving nucleic acids has been investigated for many years (for review, see ref. 20) ; in particular, different binding modes have been proposed : interaction between the ions and the phosphate © IRL Pre» Limited, Oxford, England. 4247 ABSTRACT Nucleic Acids Research groups, either d i r e c t l y or via a water bridge, interactions with electron donor sites of the bases,... (21,22). The IR spectra of sodium salt poly(dG-dC).poly(dG-dC) have been partly described (10,11,23). In the present work, we have investigated by IR spectroscopy the conformation effect of Mg + , Co and Ni on poly(dG-dC).poly(dG-dC), and the possible binding sites of these ions. The use of a polynucleotide, s p e c i f i c a l l y deuterated on the 8-carbon of guanines (poly(dD8G-dC).poly(dD8G-dC)), allows to localize a s t r u c t u r a l l y important interaction of the t r a n s i t i o n metal ions with the N7 s i t e of this base. Poly(dG-dC).poly(dG-dC) ( l o t 719-76), 2'-deoxyguanosine 5'-phosphate (dGMP) and neutral 2'-deoxycytidine (dC) were purchased from P.L. Biochemicals. Specific deuteration of the guanines was obtained by incubation in D20 at 80°C f o r 3 hours (24-25). Samples were deposited on ZnSe windows and gently dried so as to give homogeneous f i l m s . The desired amount of ions is obtained by diffusion of a droplet of the corresponding solution (NiClp, CoBr-, MgCl 2 , at pH 7 ) , followed by slow evaporation. Films are placed i n cells with controlled r e l a t i v e humidity (H20 or D20) (between 32 and 100* RH). The hydration of the complexes is determined d i r e c t l y on the IR spectra of the samples. Ion/nucleottde ratios were varied between 1 and 5. The Perkin-Elmer 180 double-beam spectrophotometer is coupled to a Hewlett-Packard 9825 A calculator allowing systematic data treatment such as baseline and water contribution corrections, scaled spectrum substract1on,... RESULTS Z conformation of poly(dD8G-dC).poly(dD8G.dC) in the presence of divalent ions At high RH (> 76*), and whatever the ions (Co 2 + , N i 2 + , Mg 2+ ), the I.R. spectra r e f l e c t a Z type double helix conformation of the polynucleot i d e . The spectral regions corresponding to the absorpttons of the bases, of the sugar-base glycosidic linkage and of the phosphodiester backbone show the characteristic features of the left-handed structure. In the 1750 cm^-lSSO cm"1 region, the drastic modification of the base pair stacking which occurs during the B •+• Z t r a n s i t i o n induces Important s h i f t s of the bands corresponding to the double bond vibrations 4248 MATERIALS AND METHODS Nucleic Acids Research Abs. Ha 1600 Figure 1. Infrared spectra of poly(dD8G-dC).poly(dD8G-dC) in D2O In the base double bond stretching vibration region2 + : a/ Na + B form ; b/ Na , 2+ Z form ; c/ Ni2+ (Or Co2+, or M g ) , 1 M per nucleotide, Z form. of the bases. In D 2 0, two bands are observed 1n the poly(dD8G-dC). poly(dD8G-dC) - Na+ spectrum, located at 1681 cm"1 and 1649 cm"1 in the B conformation (Fig. la). They are found respectively at 1666. cm" and 1634 cm in the Z conformation (Fig. lb). In the presence of divalent metal ions, the spectrum recorded in D2O shows two absorptions at the same latter wavenumbers (F1g. lc). In the case of spectra recorded in H-0, the modification of the base pair stacking in the sodium salt polymer is reflected by a shift of the 1710 an" 1 band to 1692 cm"1 (Fig. 2). In the presence of divalent ions, this absorption is also detected at 1692 cm" (results not shown). Thus, the absence of the 1710 cm" band (in HUO), and the presence of a doublet at 1666 an" -1634 an (in D~0) can be correlated to a Z type basa stacking of the poly(dD8G-dC).poly(dD8G-dC) in the presence of divalent metal ions. In the 1550 cm"1-1250 an" 1 region (Fig. 3 ) , that is to say in the spectral region corresponding mainly to the in plane vibrations of the 4249 1700 B forir Nucleic Acids Research Aba. 1600 1400 1200 1000 Figure 2. IR spectra of poly(dD8G-dC).poly(dD8G-dC).Na 98* RH), top : Z form (86X RH). : bottom : B form Aba. 1500 1400 Figure 3. IR spectra of poly(dD8G-dC) .poly(dD8G-dC) in the sugar base and glycosidic linkage vibration region. Ion/nucleotide ratio •> 1. RH > 76t ; a/ Na+, b/ Mg 2 + ; c/ N12+ ; d/ Co 2 + . 4250 1800 Nucleic Acids Research Abs. 900 800 Figure 4.IS spectra of out of plane vibrations of poly(dD8G-dC).poly(dD8G-dC) in O2O : a/Na+, B form ; b/ Co2+ (1 per nucleotide), Z form ; c/ Ni?+ (1 per nucleotide), Z form. bases, the spectra of the poly(dD8G-dC).poly(dD8G-dC)-M2+ are characterized by the presence of the 1409 c m , 1321 cm"1 and 1264 cm"1 bands, which are also (...truncated)