Zinc finger proteins are essential for the proper functioning of eukaryotic organisms, while their artificial variants can be used in cancer therapy or gene editing via their specific DNA recognition. Zn(II) binding is indispensable for the structural integrity of zinc fingers. While metal ion binding of zinc fingers has been extensively investigated in vitro, intracellular studies are rather scarce. Herein, we utilized the novel I-Block assay, based on the regulation of β-galactosidase expression via the transcription inhibition ability of zinc finger proteins inside the modified ER1821ΔLacI Escherichia coli cells. The results indicated that the performance of the assay largely depends on the position of the DNA sequence targeted by the zinc finger protein, but once optimized, specific DNA binding of even the rather small three-membered 1MEY# artificial zinc finger protein can be detected without large fusion tags. By fusion of 1MEY# with the nuclease domain of FokI restriction endonuclease leading to an artificial zinc finger nuclease, we demonstrated that the I-Block system can distinguish between DNA binding and DNA cleavage inside the cells allowing for zinc finger nuclease screening. The investigation of metal ion/metalloid-zinc finger protein interactions within cellular conditions provided direct proof for the inhibitory effect of Cd(II), Hg(II), and As(III) on the DNA binding of a zinc finger protein at a molecular level.
Hajdu et al. (Tue,) studied this question.