Monovalent Copper and Silver Ions Block DNA Polymerase Chain Reaction

In the present study we present the dramatic effect that monovalent copper ions (Cu(I))have on the DNA polymerase chain reaction, and the moderate effect which monovalent silver ions (Ag(I)) have on it. Our research utilizes the commercial Polymerase Chain Reaction (PCR) system: in anaerobic conditions, in the presence of less than 0.1  M of Cu(I) ions or in the presence of less than 10  M of Ag(I) the 11 PCR system was entirely down. 12 Under the same conditions, 1  M of divalent copper ions (Cu(II)) ions only a 13 minor effect, while10  M of divalent Ni and Zn ions shows at 14 This finding can give some explanation for the antimicrobial activity of 15 monovalent copper ions (Cu(I))as well as Although mechanism that under the 17 an unfavorable carbon molecular oxygen 18 concentration and elevated temperatures, the antibacterial action Cu(I) ions is 19 boosted, with a 10 6 bacterial population eliminated in less than 1 by 0.4mM of 20 Cu(I). Microscopy checking of E.coli morphology and scattering testes showed 21 mortality of bacteria with almost no lysis. These results suggest rapid and lethal 22 metabolic damage is the main


Introduction 33
Copper (Cu) is an essential trace element for all living organisms; in high 34 concentrations , however, it can exert a biocidal effect. Recently [1], we suggested 35 that monovalent copper ions(Cu(I)) are the active factor in copper's antimicrobial 36 activity [1]. Although the mechanism of Cu(I)'s antimicrobial effect is not yet fully 37 understood, we showed that in conditions of acidic pH, an unfavorable carbon source 38 and elevated temperatures boost the antibacterial action of Cu(I) ions. In less than 1 39 min, 0.4mM of Cu(I) eliminated a10 6 bacterial population; microscope morphology of 40 E.coli showed mortality of bacteria with almost no lysis [2]. 41 Silveris also known as abiocidal agent: especially monovalent silverions (Ag(I)) 42 [3].Copper and silver elements have a similarity as coins elements and their 43 monovalent ions have a similar electronic configuration (d 10 ). It is reasonable to 44 assume that both ions have a similar biocidal mechanism. 45 Copper ions (Cu(II) and Cu(I)) are known to form reactive oxygen species (ROS) [4] 46 that can damage bio molecules, including DNA and chromatin. This has been well-47 demonstrated in vitro with isolated DNA or chromatin, or by exposure of 48 cultured mammalian cells to copper complexes with various agents [5,6].In vivo, 49 however, according to the literature, copper ions do not catalyze the formation of 50 oxidative DNA damage [7,8,9]. 51 That said, in living cells there are mechanisms to control the intracellular 52 concentrations of copper ions [9]. ATP7b and CopApump excess copper out of the 53 cytosol and into the periplasm [10]. Once in the periplasm, copper is subject to two 54 other systems, CueO and CusCFBA, that assist CopA in controlling intracellular 55 copper levels. CueO is a multi-copper oxidase that converts Cu(I) to Cu(II), a less-56 toxic form [11].It seems that copper ions, especially Cu(I), are a major threat to the 57 cell. 58 In aqueous solutions, copper in the monovalent state (Cu + , cuprous) is unstable 59 compared to the common oxidation state of copper ion, its divalent state (Cu 2+ , 60 cupric). Cu + (aq) will disproportionate to Cu 2+ (aq) and Cu 0 (s);it rapidly reacts with 61 molecular oxygen, and as a consequence, Cu + (aq) concentrations are usually very low. 62 To achieve significant concentrations of Cu + ions, a ligand, such as Acetonitrile [12], 63 benzoic acids [13] and ATP [14] has to be added to the solutions. The ingredients are assembled in a tube along with the cofactors needed by the 100 enzyme, and are put through repeated cycles of heating and cooling that allow the 101 DNA to be synthesized. 102 In this study all the PCR experiments went through the following stages: (1)  by restriction enzymes) that were incubated with 8MCu(II), Ni(II), Zn(II) and Cu(I) 120 in anaerobic atmosphere at the PCR conditions as described before. The segments 121 tested for changes on Gel electrophoresis technique. Cu(I) to inhibit the Taq polymerase. In all examined ranges of concentration, the 137 DNA production was blocked (in 0.8M some activity was observed but in lower 138 concentrations; at 0.08M no activity was detected). Allowing molecular oxygen to 139 interfere decreases the effect of Cu(I) and at a saturation of lower than 8M copper 140 ions, the DNA amplification appear. 141 It is reasonable to assume that most of the Cu(II) effect is a consequence of partial  In this study we showed that very low concentrations of monovalent copper 175 ions(1*10 -7 M Cu + ) and low concentrations of monovalent silver ions (1*10 -5 MAg + ) 176 cause a complete deactivation of DNA polymerase.
This provides some clues about the mechanism of the bactericidal effect of Cu + and 178 Ag + previously described by our group (1).The mechanism is not one of oxidative 179 stress, even though monovalent copper ions (much more than bivalent iron ions) react 180 with molecular oxygen (do not need hydrogen peroxide as do bivalent copper and iron 181 ions) to generatere active oxygen species (ROS), so that a Fenton-like reaction can 182 begin without hydrogen peroxide [4]. If the mechanism was one of oxidative stress, 183 we would expect that an aerobic atmosphere would enhance the effect. But the results 184 (Fig 1 compared to Fig 2) show the inverse: an anaerobic atmosphere increases the 185 effect. Copper ions, both monovalent and bivalent, do indeed generate reactive 186 oxygen species (ROS) but the impact of the latter on the system is negligible 187 compared to the main effect. Monovalent silver ions do not generate reactive oxygen 188 species (ROS) and yet Ag + has a bactericidal effect, apparently via a mechanism 189 similar to that of Cu + . 190 Research articles that have examined the role of oxidative stress generated by copper 191 ions found no damage to DNA in vivo [7,8,9]. At non-cytotoxic concentrations, 192 copper ions inhibit the repair of oxidative DNA damage induced by visible light [8], 193 the inhibition probably resulting from damage to enzyme function caused by Cu(I) 194 rather than from oxidative stress. Figure 6 shows that the DNA plasmids maintain 195 their weight and are not harmed by monovalent copper ions and molecular oxygen. 196 In fact, Cu(I) can serve as an antioxidant agent. Recent results [16] show that a Cu(I) 197 complex with ATP reacts very rapidly with a methyl radical (CH3 . ) to produce Cu(II) 198 and methane, terminating the radical chain: 199 3.

202
Indeed, there is a report [8] that shows that copper ions reduce the oxidative DNA 203 damage caused by Fe(II) and H2O2. 204 Copper(I) ions do not undergo the disproportion reaction in the PCR medium that is 205 expected in aqueous solutions. In our research we added Acetonitrile to the medium 206 as a stabilized ligand. Recent results [16] show that ATP and Adenosine form a 207 strong complex with copper(I) ions, and shift the disproportion reaction to the left: 208 4. 2Cu + ATP Cu +2 + Cu 0 209 210 In vivo it is reasonable to assume that ATP and other nucleic acids serve as stabilized 211 ligands for Cu(I) inside the cell. Calculations [16] suggest that in the complex Cu(I)-212 ATP, the Cu(I) interacts with the base (adenine) and the phosphate groups causes the 213 ATP to distort and fold, probably disrupting its function as a co-factor in the 214 enzymatic system. 215 Ions such Zn +2 and Ni +2 have a negligible effect on the system;Cu +2 have some effect 216 ( fig 5). It is known that the system needs Mg +2 ions for proper operation of the 217 enzyme, but it does not seem that in the tested concentrations, the partial exchange of 218 Mg +2 ions by Zn +2 and Ni +2 or Cu +2 has a significant effect. It is possible that all of the 219 effect of Cu +2 is due to a reduction to Cu + in a stabilized Cu + environment. It was mentioned in the literature that Cu(II) is rapidly reduced to Cu(I) by sulfhydryl 221 ligands in solution, including glutathione and cysteine (17). 222 Previous article [1] demonstrates that Cu + is in two order of magnitude a more potent 223 antimicrobial agent than Ag + . In this study we founds that Cu + bloke DNA 224 polymerase chain reaction with concentration low in two order of magnitude from 225 Ag + concentration (fig 4), it support the assumption that the Cu(I) and Ag(I) anti-226 bacterial mechanism functions via enzymatic inhibition 227 The assumption that the Cu(I) anti-bacterial mechanism functions via enzymatic 228 inhibition is also supported by recent results [2] showing that elevated temperatures 229 boost the antibacterial action of Cu(I) ions. We suggest that Cu + and Ag + acts by interfering with enzymatic metabolisms, such 248 asDNA polymerase, via uncompetitive inhibition of the enzymes. 249 More relevant than ever this finding may suggest a possible role of Cu(I) producing 250 system as an antiviral agent as well. However that concept is yet to be proven. 251 252

Conclusions 253
Our result show the dramatic influence of Cu(I) and Ag(I) on the replicative ability of 254 DNA. Zn(II), Ni(II) and Cu(II) have minor effects compared to Cu(I)and Ag(I). 255 Figure 1 Example of gel-electrophoresis results of PCR experiments inanaerobic conditions. Normal Solution Composition (NSC) was used, with additions of Cu(II) and Cu(I) ions at nal concentrations of between80 and0.08µM.

Figure 2
Example of gel-Electrophoresis results of PCR experiments in aerobic conditions. . Normal Solution Composition (NSC) was used, with additions of Cu(II) and Cu(I)ionsat nal concentrations of between80 and0.08µM.

Figure 3
Gel-electrophoresis results of PCR experiments. Ag(I)ions added at nal concentrations from 80 to 0.08µM.

Figure 5
Gel-electrophoresis results of DNA segments (from plasmids cut by restriction enzymes) that were incubated with 8µMCu(II), Ni(II), Zn(II) and Cu(I) under aerobic conditions.