Metal–DNA interactions

Journal of Molecular Structure 651–653 (2003) 19–26 www.elsevier.com/locate/molstruc

Metal –DNA interactions Jane Anastassopoulou* National Technical University of Athens, Chemical Engineering Department, Radiation Chemistry and Biospectroscopy, Zografou Campus, 15780 Zografou, Athens, Greece Received 15 October 2002; revised 8 November 2002; accepted 8 November 2002

Abstract Metal ions can bind to DNA directly or indirectly through hydrogen bonding of the coordinating water molecules surrounding the metal ions. Metal binding to the bases usually disrupts base pair hydrogen bonding and destabilizes the double helix. Spectroscopic and X-ray data shows that the N7 atom of the purine or N3 of pyrimidine residues as well as exocyclic O atoms and the phosphate oxygen atoms are the preferential sites of metal binding. The binding of metals to DNA and RNA also influences indirectly the sugar conformation. As a result of this change in sugar puckering the two helical conformations of ADNA and B-DNA or RNA are characterized by the orientation of the bases with respect to the axis of the double helix. This may be the reason why some metal ions influence DNA synthesis and the replication process. In this paper we describe the influence of metal ion binding on the DNA structure and the variation of binding behaviour of different metal ions. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Metal ions; DNA; RNA; Metal–DNA complexes; Structures; FT-IR spectra

1. Introduction It is well established that organic life depends on inorganic elements for carrying out many vital processes [1,2]. Life metal ions play a crucial role in the human body and small deviations from normal levels of concentration are recognized as symptoms of malfunctions or diseases [3]. They are essential for several cell reactions and varied metabolic and physiological functions. No one can deny the importance of Naþ, Kþ, Mg2þ and Ca2þ ions in cells and as neurotransmitters [4,5]. Trace and ultratrace metal ions control essential biological processes of living cells and without their catalytic * Corresponding author. Tel.: þ30-210-7723133; fax: þ 30-2107723184.

presence many biological reactions would not take place. The appearance of several diseases may be related to metal ion depletion. For instance, deficiency of iron, magnesium or calcium causes anaemia, cardiovascular diseases or osteoporosis, respectively [6]. However, they become toxic to cells when their concentrations surpass certain optimal (natural) levels. When there is excess of metals, such as, Cu(II) and Fe(II) in Wilson’s and thalaseamia diseases, correspondingly, then chelating agents may be used to reduce their concentration [7 –9]. On the other hand, the toxicity of heavy metal ions may depend partly on their binding to specific DNA sites [10 – 11]. In the last years there is an increasing number of publications concerning conformation-specific interactions of DNA with metal ions. The renewed interest in the DNA –metal ion

0022-2860/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 2 8 6 0 ( 0 2 ) 0 0 6 2 5 - 7

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interactions is due to the developed sensitive techniques and the possibility to synthesize sequence-specific oligomers, which allow crystal structure determination [12 – 14].

2. Metal ion –DNA interactions The binding of metal ions to nucleic acids has been subject of study for many years, but the mechanism of their action is still unknown. DNA is a supercoiled negatively charged polymer of nucleotide units and is found in cells usually as right-handed double helix (BDNA). The two strands (chains) have complementary sequences of nucleic bases, with the phosphate groups on the outside and the base pairs linked by hydrogen bonding in the interior. The four nucleotides are consisted of the bases adenine (A), guanine (G), thymine (T) and cytidine (C) and a sugar-phosphate group (Fig. 1). The positively charged metal ions interact directly or indirectly with sites characterized by high electron density or negatively charged residues of DNA. Such sites on DNA could be the negatively charged phosphates of the backbone of both strands and the electron donor atoms of the bases, such as N and

O. The predominant mode of metal binding takes place at the N7 and O6 of guanine and N7 and N1 of adenine bases and the N3 of pyrimidines. The possible ways of metal binding are shown in Fig. 1. Metal ions can bind tightly to DNA as partially dehydrated or fully hydrated and this binding can be direct or indirect with DNA. There is evidence that metal ions interact also between these two extremes. Metal ions exist as ‘free’ or ‘bound’ in the body. They are ‘free’ hexahydrated when they can move around in the body liquids and they are ‘bound’ when they form complexes with covalent bonds. Let us examine the various metals of the Periodic Table and their interactions with DNA.

2.1. Alkali metals The monovalent alkali metals are extremely reactive and occur in nature only as cations. The alkali metals can produce electrostatic or ionic binding and the binding depends on their electronic structure. With some exceptions, alkali metals interact in an outer sphere manner. They do not bind tightly to DNA. It is known that both metal ions and nucleic acids are specifically hydrated and an overlapping of

Fig. 1. DNA double helix. (a) The complementary A –T and G –C bases are linked through hydrogen bonds, two for AT and three for GC pairs. (b) Metal ions can bind to one two sites of the same strand (intrastrand) or of the opposite strand (interstrand), or by intercalation in complex form between the bases. The binding of metal ions can lead to single strand break (ssb) or to double strand breaks (dsb).

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their hydration spheres and the release of water molecules accompany the interaction between them and the bulk state. Spectroscopic as well as crystallographic data show that alkali metals are concentrated on some reiterative DNA sequences. Recently, Danisov and Halle using Magnetic Relaxation Dispersion (MRD) for the dodecamer sequences CGCGAATTCGCG (DDD, Dickerson-Drew Dodecamer) found that Naþ binds preferentially to A-track minor groove of the oligonucleotide and are in accordance with the X-ray crystal data of DDD in the presence of Naþ ions [15,16]. Clear evidence of a single alkali metal ion bound in minor groove of DDD was provided by its crystal structure in the presence of Rbþ ion [17]. From these crystallographic data was found that Rbþ ion prefers to sit in the centre of the ApT step of the minor groove of DDD. Rbþ ion binds with an inner sphere manner to O2 atoms of thymines of the two opposite strands (interstrand binding). Monovalent metal ions prefer to interact with AT rich region of the minor groove. In order to enter in the groove as is shown below (Fig. 2) [14, 18 –20], they should release their coordinated water and interact directly with the bases. Molecular dynamic (MD) simulations, solution NMR and crystallographic results agree that the monovalent cations Naþ, Kþ, Rbþ, Csþ and NHþ 4 prefer direct binding (inner sphere) at the ApT step in A-track in minor groove of DNA [21,22]. The MD results suggest that the presence of metal ions into

the minor groove is accompanied by groove narrowing. It was also suggested by MD simulations that the geometry of Naþ coordination in the minor groove depends on the exact position of the cation [14,15].

Fig. 2. Monovalent cation in the minor groove of DNA [14].

Fig. 3. Proposed structure of Mg(H2O)5-GMP binding Mg2þ [33].

2.2. Alkaline earth metals The divalent alkaline earth metals are rather more reactive than alkali metals. They react as the alkali metals do and more. They can coordinate with mono- and bi-dentate ligands. From alkaline earth metals magnesium is the major intracellular divalent ion and is present in all DNA and RNA activation processes. Mg2þ cations can act as bridge between specific enzymes and nucleotides, nucleosides and its derivatives [23]. It was found using Fourier transform infrared and Raman spectroscopy as well as NMR spectroscopy that magnesium ions play a significant role in the stabilization of secondary and tertiary structure of the DNA [24 –33]. The two positive charges of Mg2þ ions attract the negatively charged phosphate groups and with six-coordinated water molecules form hydrogen bonds. The N7 site of the nucleotide could substitute one of the coordinated water molecules. Another one can be involved in hydrogen bonding with O6, while the others form more hydrogen bonds (Fig. 3). From FAB-MS spectra of magnesium-50 -guanosine monophosphate we found base-magnesium fragments, which suggest that the pentahydrate doubly þ charged magnesium moiety, Mg(H2O)þ 5 , is bound to the base guanine and directly to N7 site of

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Fig. 4. (A) hexahydrated magnesium cation binding to DNA, only through hydrogen bonding, (B) pentahydrated through coordinating and hydrogen bonding and (C) bridged hexahydrated, hydrogen bonded.

the guanine. Molecular simulations also showed that magnesium ions bind to nucleotide directly to N7 and indirectly to O6 and the phosphate groups (Fig. 4) [34]. The binding of the cations between N7 and phosphate oxygens is accompanied by a change in the conformation of DNA. It was shown that increasing Mg2þ cation concentration changes the B-DNA form to Z-DNA [35 – 39]. The B-DNA sequence was also affected by other divalent cations, such as, Ca2þ, Zn2þ, Co2þ, Ba2þ, Mn2þ and Cd2þ [40,41]. In general, divalent cations are widely known that affect the DNA conformation (Fig. 3). We have seen that when hexahydrate magnesium ion interacts with 1-Methylcytidine (1-Mecyt) and cytidine (cyt) it produces firstly supramolecular complexes through its six coordinated water molecules, with the general chemical formula [Mg(H2O)6(L)6]þ þ , where L ¼ 1-Mecyt and cyt, forming ten hydrogen bonds between the 6 water molecules and the 6 cytidines or methylcytidines [42, 43]. By comparison of these two ligands it was found

that magnesium binds specifically to O2 and N3 sites of 1-Mecyt only through water molecules (outer sphere). However, when hexahydrate magnesium ion interacts with cyt it can replace two or four of its six water molecules with equal numbers of cyt moieties (inner sphere). In this case magnesium binds directly to O2 site of cyt, leading to inner coordinated complexes of the type [Mg(H2O)4(cyt)2]þ þ and [Mg(H2O)2(cyt)2]þ þ (Fig. 5). It is important to notice that the type of the formed coordinated complexes is time and temperature dependent. From the above results it is obvious that the type of the complex depends both on the nature of the cation and/or the ligand. FT-IR spectroscopic data show that interaction of magnesium cations with GAAGCTTC oligonucleotide give complex with adenine (A), guanine (G) and thymine (T) residues above and below G – C pairs, but not with thymine moiety, as it has resulted from the spectra Fig. 6. Considerable reduction in intensity of the bands at 1690 and 1450 cm21 is observed as the ratio M:L increases from 1:1 to 6:1. These results are

Fig. 5. The molecular structures of magnesium nucleic bases: complexes, (A) inner coordinated complexes. [Mg (H2O)2 (cyt)4]þ þ and (B) hydrogen bonded [Mg(H2O)6 (1-Mecyt)2]þ þ super molecule.

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Fig. 7. Structure of [Co(H2O)6(1-Mecyt)6]2þ ion. Cobalt binds through hydrogen bonds to O2 and N3 sites of 1-Mecyt.

Fig. 6. FT-IR spectra in the region 2000– 1400 cm 2 1: (L) GAAGCTTC (uncomplexed) double helix and the (C) [GAAGCTTC-Mg(H2O)5] complex 1:1 [45].

in agreement with the results obtained using ultrasonic and densimetric techniques. [44]. From the FT-IR spectra in combination with X-ray and NMR data we proposed that when Mg2þ interacts with oligonucleotide GAAGCTTC it must bind at the terminal GpA and GpC. The binding sites are the electronegative PO2 2 groups, the CyO, NH2, N1, N3 and N7 of the base residues [45,46]. From the alkaline earth metals Ca2þ, Sr2þ and 2þ Ba usually interact with DNA in an inner sphere manner, Mg2þ is engaged in more outer-sphere complexes, because of its stability and higher Gibbs free energy of hydration [47,48]. 2.3. Transition metals Transition or d-block metals have the d-orbitals partially filled and thus they can qualify as free radicals. Transition metals interact with more than two different sites and their interactions with DNA are more complicated. Transition metals loose their water molecules very easily and give inner sphere coordinated complexes [49]. They usually

bind directly to the bases and indirectly to the phosphate groups. Fig. 7 shows the crystal structure of [Co (H2O)6(1-Mecyt)6]þ þ complex [50]. As in the case of magnesium ions 1-Mecyt interacts in an outer sphere manner with the cobalt ion. The same structure was also observed with Mn2þ ions [50]. FT-IR spectra have shown that Mn2þ cations form zwitterionic type complex with 50 -guanosine monophosphate, by replacing the sodium cations of the phosphate group [51]. Most of the transition metals react chemically with the N7 atom of purine or N3 of pyrimidine and perturb the double helix. The binding of transition metal particular at G – C sites of DNA leads to its damage through radical generation from oxidation by H2O2 [52 –54] (Fig. 1). Among the post transition metals zinc plays an important role in stabilization of the finger-loop domain in DNA-binding proteins [55]. In order to find the specific interactions between Zn2þ and 50 guanosine monophosphate (50 -GMP) we synthesized complexes in various molar ratios [Zn]:[50 -GMP]. From the ‘marker band’ at 822 cm21 of the FT-IR spectra, which corresponds to C20 -endo sugar conformation it was suggested that Zn2þ cations form 50 GMP complexes, which depend on the concentration of the cation [56]. Thus it was observed starting from the ratio 1:1 that the metal ion binds to N7 site of the base and the sugar conformation is C30 -endo(/). Increasing the ratio [Zn]:[50 -GMP] . 4:1 the sugar conformations C30 -endo (/)and C20 -endo exist in a ratio 50:50 [57]. From the above data it is concluded

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Fig. 8. A Zn2þ ion binds at four phosphate oxygens and two water molecules.

that Zn2þ cations form two different complexes, where in one Zn2þ binds to oxygen atoms of the phosphate group, while in the other it binds to N7 atom of the base. Recent crystal structure of Zn –DNA complexes shows that a Zn2þ has a tendency to bind to four oxygens of four different phosphates, while another Zn ion binds to N7 site of the guanine base, in agreement with our spectroscopic data (Fig. 8) [58]. Further investigation showed that the formed complexes, as well as their hydration, depend on the anion of the Zn2þ salts. Thus, the complex that

was produced from the reaction of Zn(NO3)2 – 6H2O was crystallized with two water molecules and the sugar conformation was found to be C30 endo, supporting the binding of Zn to N7 site of the base (Fig. 9). On the other hand, the complex that was produced from ZnSO4 salt is crystallized with four water molecules and shows C20 -endo conformation [59]. This observation leads to the result that we have to take into consideration of the kind of the anions of the metal salts that we use for the interaction, which also play a role in the complex formation.

3. Metal –DNA site binding The conformation of nucleic acids depends on the kind of metal ion that binds to DNA. Metal binding to the bases will usually disrupt base pare hydrogen bonding and destabilize the double helix [60,61]. On the other hand metal ions neutralize the excess negatively charged phosphate groups and this stabilizes the helix. The binding of metals to nucleotides or polynucleotides does influence the sugar conformation [62]. As a result of this change in sugar puckering two helical conformations of B-DNA, A-DNA are formed together with Z-DNA or RNA, which are characterized by the orientation of the bases with respect to the axis of helix. Thus, in B-DNA all sugars are in C20 -endo puckering, in A-DNA all sugars are in C30 -endo puckering and in water solutions there is an equilibrium C20 endoˆ!C30 -endo 50:50. In Table 1 are shown the preferential sites of metal binding to DNA. Furthermore, in the minor groove cations localize preferentially at AT-rich sequences, while in the major Table 1 Metal–DNA binding sites

Fig. 9. The influence of anions on the metal-mononucleotide binding of the complex Zn-5 0 -GMP: (a) 5 0 -GMPNa2 , (b) Zn(NO3)2·6H2O, (c)ZnSO4·7H2O, (d) ZnCl2. FT-IR spectra [59].

Binding site

Metal

Base Phosphate group Phosphate and base

Agþ, Hgþ, Pt2þ Liþ, Naþ, Kþ, Rbþ, Csþ, Mg2þ, Ca2þ Sr2þ Ba2þ Cr3þ, Fe3þ Co2þ, Ni2þ, Mn2þ, Cd2þ, Pb2þ, Cu2þ, Fe2þ, Mg2þ, Fe3þ

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groove the preferential site is the GC-rich sequences. The binding of metal ions to DNA is sequence dependent [13,63].

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