METALLOTHIONEIN PROTECTS DNA FROM COPPER-INDUCED BUT NOT IRON-INDUCEDCLEAVAGE IN-VITRO

Iron and copper ions mediate generation of reactive oxygen radicals fr om O-2 and H2O2 by the Fenton reaction: these radicals are capable of damaging DNA. We studied (a) the ability of these metals to induce dou ble-strand breaks in DNA in vitro in the presence of H2O2 and ascorbic acid as donors of reactive oxygen, and (b) the ability of the metal-b inding protein metallothionein (MT) to protect DNA from damage, Strand cleavage was measured by loss of fluorescence after binding to ethidi um bromide and by increased mobility of DNA in agarose. The results sh ow that Cu(II), Fe(II) and Fe(III) all can induce damage to calf thymu s DNA under our experimental conditions. Cu(II)-induced DNA damage was dose-dependent and the degree of damage was proportional to the conce ntration of H2O2. other hand, DNA fragmentation was significant only i n the presence of high concentrations of Fe(II) or Fe(III), Addition o f Zn-MT to the reaction mixture prior to addition of Cu(II) inhibited fragmentation of DNA in a dose-dependent manner but had little effect on iron induced damage. Other proteins (histone or albumin) were not e ffective in protecting DNA from Cu-induced damage, as compared to Zn-M T. The formation of Cu(I) from Cu(II) in the presence of hydrogen pero xide and ascorbate was also inhibited by addition of Zn-MT. Thus, MT m ay protect DNA from damage by free radicals by sequestering copper and preventing its participation in redox reactions.