Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.

Chromium(VI) salts are well known to be mutagens and carcinogens and to easily cross the cell membranes. Because they are powerful oxidizing agents, Cr(VI) reacts with intracellular materials to reduce to trivalent form, which binds DNA. This study was designed to investigate the interaction of calf thymus DNA with Cr(VI) and Cr(III) in aqueous solution at pH 6.5-7.5, using Cr(VI)/DNA(P) molar ratios (r) of 1:20 to 2:1 and Cr(III)/ DNA(P) molar ratios (r) of 1:80 to 1:2. UV-visible and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the metal ionbinding sites, binding constants, and the effect of cation complexation on DNA secondary structure. Spectroscopic results showed no interaction of Cr(VI) with DNA at low anion concentrations (r ‫؍‬ 1:20 to 1:1), whereas some perturbations of DNA bases and backbone phosphate were observed at very high Cr ( Chromium(VI) salts are well known to be mutagens and carcinogens and to easily invade the insides of cells (1). Cr(VI) produced DNA cross-links in rat tissues in vivo (2) and in cultured cells in vitro (3, 4). Although Cr(VI) damaged nuclear DNA in whole cells, no reaction of Cr(VI) with isolated DNA occurred in vitro at physiological pH in the absence of a metabolizing system (5). The Cr(VI) that is taken up is considered to be reduced by glutathione, cysteine, or ascorbic acid into Cr(III) (6), and the resulting cation reacts with DNA to form Cr(III)-DNA adducts. Because Cr(III) is a final form of chromium within the cell, the interaction of Cr(III) with DNA may play crucial role in the carcinogenetic action of Cr(VI) salts.The conversion of B form into Z form in the purine-pyrimidine sequence of DNA has been considered to be a factor in the transcriptional activity of genes (7). Cr(III) is found to interact with the poly(dG-dC) at low concentration and change B form to Z form in the presence of ethanol (8). However, Cr(III) at high concentration causes DNA condensation, inhibiting the alteration of B to Z structure (8). Moreover, the study on the effect of Cr(III) on DNA replication with single-stranded DNA template and micromolar concentration of Cr(III) revealed that Cr(III) bound in a dose-dependent manner to the template DNA and prevents DNA replication (9). However, if the unbound chromium was removed from the system by gel filtration, the rate of DNA replication by polymerase I (Klenow fragment) on the chromium-bound template increased more than 6-fold relative to control. This increase was paralleled by as much as a 4-fold increase in processivity and a 2-fold decrease in replication fidelity. When the concentration of Cr(III) increased further, DNA-DNA cross-links occurred to inhibit the polymerase activity. Trivalent chromium can bind purified DNA and form lesions capable of obstructing DNA replication in vitro (10, 11). It has also been observed that intact Novikoff ascites hepatoma cells exposed to potassium chromate formed cross-linking of nuclear proteins to DNA (12). Recently, Cr(III) was shown to cause mutation...

Although structural differences for the Mg-DNA and Ca-DNA complexes are provided in the solid state, such comparative study in aqueous solution has been less investigated. The aim of this study was to examine the bindings of Mg and Ca cations with calf thymus DNA in aqueous solution at physiological pH, using constant concentration of DNA (1.25 or 12.5 mM) and various concentrations of metal ions (2 microM-650 microM). Capillary electrophoresis, UV-visible, and Fourier transform infrared spectroscopic methods were used to determine the cation-binding modes, the binding constants, and DNA structural variations in aqueous solution. Direct Ca-PO(2) binding was evident by major spectral changes (shifting and splitting) of the backbone PO(2) asymmetric stretching at 1222 cm(-1) with K = 4.80 x 10(5) M(-1), whereas an indirect Mg-phosphate interaction occurred (due to the lack of shifting and splitting of the phosphate band at 1222 cm(-1)) with K = 5.6 x 10(4) M(-1). The metal-base bindings were directly for the Mg with K = 3.20 x 10(5) M(-1) and indirectly for the Ca cation with K = 3.0 x 10(4) M(-1). Both major and minor groove bindings were observed with no alteration of the B-DNA conformation.

The involvement of the Fe cations in autoxidation in cells and tissues is well documented. DNA is a major target in such reaction, and can chelate Fe cation in many ways. The present study was designed to examine the interaction of calf-thymus DNA with Fe(II) and Fe(III), in aqueous solution at pH 6.5 with cation/DNA (P) (P = phosphate) molar ratios (r) of 1:160 to 1:2. Capillary electrophoresis and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the cation binding site, the binding constant, helix stability and DNA conformation in Fe-DNA complexes. Structural analysis showed that at low cation concentration (r = 1/80 and 1/40), Fe(II) binds DNA through guanine N-7 and the backbone PO(2) group with specific binding constants of K(G) = 5.40 x 10(4) M(1) and K(P) = 2.40 x 10(4) M(1). At higher cation content, Fe(II) bindings to adenine N-7 and thymine O-2 are included. The Fe(III) cation shows stronger interaction with DNA bases and the backbone phosphate group. At low cation concentration (r = 1:80), Fe(III) binds mainly to the backbone phosphate group, while at higher metal ion content, cation binding to both guanine N-7 atom and the backbone phosphate group is prevailing with specific binding constants of K(G) = 1.36 x 10(5) M(-1) and K(P) = 5.50 x 10(4) M(-1). At r = 1:10, Fe(II) binding causes a minor helix destabilization, whereas Fe(III) induces DNA condensation. No major DNA conformational changes occurred upon iron complexation and DNA remains in the B-family structure.

The question addressed in this study is how does the protein-DNA complexation affect the structure and dynamics of DNA and protein in aqueous solution. We examined the interaction of calf-thymus DNA with human serum albumin (HSA) in aqueous solution at physiological conditions, using constant DNA concentration of 12.5 mM (phosphate) and various HSA contents 0.25 to 2% or 0.04 to 0.3 mM. Affinity capillary electrophoresis and FTIR spectroscopic methods were used to determine the protein binding mode, the association constant, sequence preference, and the biopolymer secondary structural changes in the HSA-DNA complexes. Spectroscopic evidence showed two types of HSA-DNA complexes with strong binding of K(1) = 4.5 x 10(5) M(-1) and weak binding of K(2) = 6.10 x 10(4) M(-1). The two major binding sites were located on the G-C bases and the backbone PO(2) group. The protein-DNA interaction stabilizes the HSA secondary structure. A minor alteration of B-DNA structure was observed, while no major protein conformational changes occurred.

No abstract

Hexavalent chromium [Cr(VI)] is a known etiological factor in human lung cancer. Cr(VI) exposure-related lung cancer has a high mutation incidence in the p53 gene. Upon intake in human cells Cr(VI) is reduced to Cr(III), which is able to conjugate with amino acids and consequently form either binary Cr(III)-DNA or ternary Cr(III)-amino acid-DNA adducts. Both binary and ternary Cr(III)-DNA adducts are mutagenic. We have found that the Escherichia coli nucleotide excision enzyme UvrABC nuclease is able to incise Cr(III)- and Cr(III)-histidine-modified plasmid DNA and the extent of incision is proportional to the amount of Cr(III)-DNA adducts in the plasmid. In order to determine the role of Cr(III)-DNA adducts in the mutagenesis of the p53 gene in human cancer using the UvrABC nuclease incision method, we have mapped the Cr(III)-DNA distribution in PCR DNA fragments amplified from exons 5, 7 and 8 of the p53 gene. We have found that the sequence specificities of Cr(III)-DNA and Cr(III)-histidine-DNA adducts in the p53 gene sequence are identical and that both types of adducts are preferentially formed at -NGG- sequences, including codons 245, 248 and 249, the mutational hotspots in human lung cancer. It has been found that Cr(III)-DNA adducts induce mainly G to T mutations. Therefore, these results suggest that Cr(III)-DNA adduct formation contributes to the p53 gene mutations in lung carcinogenesis.

Chromium (VI) [Cr(VI)], a ubiquitous environmental carcinogen, is generally believed to induce mainly mutagenic binary and ternary Cr(III)-deoxyguanosine (dG)-DNA adducts in human cells. However, both adenine (A) and guanine (G) mutations are found in the p53 gene in Cr exposure-related lung cancer. Using UvrABC nuclease and formamidopyrimidine glycosylase (Fpg), and ligation-mediated PCR methods, we mapped the distribution of bulky DNA adducts (BDA) and oxidative DNA damage (ODD) in the p53 gene in Cr(VI)-treated human lung cells. We found that both BDA and ODD formed at 2'-deoxyadenosine (dA) and dG bases. To understand the causes for these Cr-induced DNA damages, we mapped the distribution of BDA adducts and ODD in the p53 gene DNA fragments induced by Cr(III), Cr(VI) and Cr(V), the three major cellular Cr forms. We found that (i) dA at -CA- is a major Cr(VI) binding site followed by -GG- and -G-. Cr(VI) does not bind to -GGG-, (ii) Cr(VI)-DNA binding specificity is distinctly different from the Cr(III)-DNA binding in which -GGG- and -GG- are preferential sites, (iii) Cr(V) binding sites include all of Cr(VI) and Cr(III)-DNA binding sites and (iv) Cr(VI) and Cr(V) induce Fpg-sensitive sites at -G-. Together, these results suggest that Cr(VI) induction of BDA and ODD at dA and dG residues is through Cr(V) intermediate. We propose that these Cr(VI)-induced BDA and ODD contribute to mutagenesis of the p53 gene that leads to lung carcinogenesis.