IN-VIVO AND IN-VITRO IRON-REPLACED ZINC-FINGER GENERATES FREE-RADICALS AND CAUSES DNA-DAMAGE

The estrogen receptor (ER) is a ligand-activated transcription factor whose DNA-binding domain (ERDBD) has eight cysteines, which coordinate two zinc atoms, forming two zinc finger-like structures. We demonstra te the capability of iron to replace zinc in zinc finger (hereby refer red to as iron finger) both in vivo (using Escherichia coil BL21 (DE3) ) and in vitro. Iron has the ability to substitute for zinc in the ERD BD as demonstrated by mobility shift and methylation interference assa ys of iron finger, which show specific recognition of the estrogen res ponse element, The DNA binding constants for both in vivo and in vitro iron-replaced zinc fingers were similar to that of the native finger. Atomic absorption analysis revealed a ratio of 2:1 iron atoms/mol of ERDBD protein, as found for zinc in the crystal structure of native ER DBD. More importantly, we demonstrate that iron finger in the presence of H2O2 and ascorbate generates highly reactive free radicals, causin g a reproducible cleavage pattern to the proximate DNA, the estrogen r esponse element. The deoxyribose method, used to detect free radical s pecies generated, and the resultant cleaved DNA ends, caused by iron f inger, suggest that the free radicals generated are hydroxyl radicals. Due to the close proximity of the zinc finger to DNA, we postulate th at iron-substituted zinc finger may generate free radicals while bound to genetic regulatory response elements, leading to adverse consequen ces such as iron-induced toxicity and/or carcinogenesis.