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DNA Interaction with Zinc(II) Ions

Zinc is a biogenic element that plays an important role in an organism. In addition to the fact that zinc is involved in the synthesis of proteins and DNA [1], it has been shown to be essential to stabilize the structure and thus the function of a large number of biomolecules including enzymes. It has been established that zinc is involved in more than 300 enzymatic reactions [2, 3]. It occurs in all tissues and fluids in relatively high concentrations. Almost 85 % of total zinc occurs in muscle and bone tissues, 11% in the skin and the liver and the rest of zinc in other tissues. The average amount of zinc in a body of an adult is about 1.4–2.3 g [4, 5]. Zinc is, after iron, the second most widely occurred transition metal in organisms [6]. Zinc(II) ions are transported into cells via zinc transporters, especially ZiP1 [7]. Zinc is found intracellularly in the eukaryotic organisms; 40% of total zinc is located in the nucleus, 50% in the cytoplasm, organelles, and specialized vesicles called zincosomes and the rest of zinc is bound in the cell membrane [8]. The concentration of zinc is relatively high in nucleus. It has been shown that zinc is involved in the processes of gene expression and the maintenance of gene stability by different ways. It stabilizes the structure of chromatin and thus affects the replication of DNA. In addition, zinc regulates transcription of RNA through the regulation of an activity of transcription factors and some enzymes, such as RNA and DNA polymerases [9].

The structural role of zinc(II) ions is accentuated in the stabilization of zinc finger motif [10]. It is an amino acid sequence, linked to zinc ion(II) to form a secondary structure in the shape of a finger [10]. Proteins with this motif have high affinity to DNA, thus, zinc finger directly mediates an interaction with DNA in the major groove [11]. Zinc is the main component of the zinc finger proteins, which represent the largest and the most diverse superfamily of nucleic acid-binding proteins and which play important roles in the regulation of transcription in the cellular metabolic network [12, 13].

Besides structural protein, zinc(II) ion plays important role also in synthesis of metal pool maintainers called metallothioneins [14-16]. The increased amount of zinc(II) ions causes an increased expression of apo-metallothionein (apo-MT) via metal-regulatory transcription factor 1 (MTF-1). Apo-MT is able to bind up to seven atoms of Zn(II), which can likely be transported into the nucleus through the nuclear pores (NPC)(Fig. 1). MTF-1 is reversibly bound to target DNA, thus, the binding depends on changes in an availability of free Zn(II) in the cytoplasm [17-19]. The binding of MTF-1 to the metal regulatory element (MRE) requires activation by zinc(II) ions. In conclusion, zinc(II) ions can enter the nucleus to activate expression of MT genes from a dietary source. Once MTF-1 senses free Zn(II), it adopts a reversible DNA-binding conformation. This allosteric change causes an exposure of zinc fingers, and the MTF-1 can move to the nucleus and activate gene expression by an association with the gene promoters that carry MREs [20]. Study by Jiang et al. confirmed that the mediation of MT gene expression starts by a translocation of MTF-1 protein from the cytoplasm to a nucleus and bind MREs in target genes [20]. However, whether metal ions can induce the creation of MTF-1 in the cytoplasm and how do other metal ions induce MT via MTF-1 remain to be elucidated [21]. A study of DNA–metal ion complex, known as M-DNA, in which a divalent metal ion is incorporated into the center of the DNA duplex, was firstly performed in 1993 [22]. Since that time, numerous studies that describe the formation, characteristics and application of this complex, has been published [23-37]. The biological role of the interaction of Zn(II) with proteins is relatively well known and exploited. The question is what role Zn-DNA complex has in living organisms and how does it affect the regulation of transcription and replication of DNA, or what relation Zn-DNA has in a processes of cancerogenesis. The suggested structure of Zn-DNA that is based on the nuclear magnetic resonance (NMR), circular dichroism (CD), and molecular modeling studies consists of GC and AT base pairs, in which the imino-proton of G and T is replaced by Zn(II), which results in an atomically thick “wire” of zinc ions sheathed by a DNA helix [24]. Zinc(II) ions also bind to phosphate groups of DNA and are able to influence (”destroy”) the basic structure of B conformation of DNA [38]. The preferred site for the coordination of the metal ion to the DNA is the N7 position of guanines [39]. Conformational change of ds or ssDNA after interacting with zinc(II) ions may be used in the regulation of transcription [40]. It has been found that the TGGGA sequence of the Xenopus 5S-RNA gene, which is essential for binding the transcription factor TFIIIA, has the highest affinity for zinc ions [39].

Various techniques have been applied to investigate the interactions of heavy metal ions with DNA. These techniques include Fourier transform infrared difference spectroscopy [27], Raman spectroscopy [38], and circular dichroism spectroscopy [41]. However, these techniques have high demands for special equipment which is not commonly available. The aim of this study was to study the interaction of zinc(II) ions with a fragment of DNA by UV/VIS spectrophotometry (changes in absorption signals and melting temperatures in denaturation) and gel electrophoresis (changes in the mobility of DNA fragments) after purification on a semi-permeable membrane.

Infrastrukturní vybavení pracoviště bylo uskutečněno díky Evropskému sociálnímu fondu projektu NanoBioTECell GA CR P102/11/1068.

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