Inhibition of Transcription Factor IIIA-DNA Interactions by Xenobiotic Metal Ions

Abstract

Transcription factor IIIA (TFIIIA), a cysteine-rich regulatory protein, is the prototype for the largest known superfamily of eukaryotic transcription factors. Members of the TFIIIA superfamily contain Cys2His2 zinc finger domains responsible for nucleic acid binding. Xenobiotic metal ions, which lack known biological function, were previously used as probes for the structure and function of steroid hormone receptors which contain Cys2Cys2 zinc finger domains. Structural alterations in cysteine-rich regulatory proteins by such ions in vivo might potentiate carcinogenesis and other disease processes. In the present study cadmium and other xenobiotic metal ions were used to probe the structure and function of TFIIIA. The specific interaction of TFIIIA with the internal control region (ICR) of the 5S RNA gene, as assayed by DNase I protection, was inhibited by Cd2+ ion concentrations of > or = 0.1 microM. Aluminum ions were also found to inhibit the TFIIIA-5S RNA gene interaction, albeit at higher concentrations (> or = 5 microM). Inhibition by either metal ion was not readily reversible. Other xenobiotic metal ions, such as mercury or cesium, were not found to be inhibitory under these conditions. None of these ions at the concentrations used in this study affected the ability of DNase I to digest DNA or restriction enzymes to specifically cleave DNA. Preincubation of TFIIIA bound to 5S RNA with either Cd2+ or Al3+ resulted in subsequent DNA binding upon dilution and RNA removal, whereas preincubation of free TFIIIA with the metal ions resulted in inhibition of subsequent DNA binding. Because 5S rRNA also binds the TFIIIA zinc finger domains, these results indicate that the 5S RNA bound to TFIIIA protects the protein from metal inhibition and implicates the zinc fingers in the inhibition mechanism. The nature of the footprint inhibition indicates that the N-terminal fingers of TFIIIA are affected by the metal ions. Cd2+ and Al3+ ions also inhibited the ability of TFIIIA to bind complementary single-stranded DNA and promote renaturation, as measured by Tris-phosphate agarose gel electrophoresis. This gel assay is sensitive to DNA conformation and Al3+ ions were found to alter the conformation of single- and double-stranded DNA in this assay. The inhibition of TFIIIA function in vitro by xenobiotic metals offers new insights into the structure and function of TFIIIA and TFIIIA-type zinc finger proteins. Inhibition by Cd2+ occurs at much lower concentrations than previously observed with steroid hormone receptors and suggests that Cys2His2 zinc finger proteins may be especially sensitive to such agents in vivo.

Assessment of occupational exposure to welding fumes by inductively coupled plasma‐mass spectroscopy and by the alkaline Comet assay

“…Besides inducing ROS, Al inhibits DNA repair enzymes that contain zinc finger domains [Hanas and Gunn, 1996]. Cui et al [2003] recently demonstrated that Al impairs the ligase step of DNA repair.…”

Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes

“…A number of reports indicate that foreign metal ions can replace Zn 2+ in various zinc finger proteins and, in so doing, impair or alter their functions [20][21][22][23][24][25][26][27]. For example, exposure to Cd 2+ inhibits the binding of transcription factors such as TFIIIA and MTF-1 to their cognate DNA sites [19,27].…”

“…For example, exposure to Cd 2+ inhibits the binding of transcription factors such as TFIIIA and MTF-1 to their cognate DNA sites [19,27]. In contrast, another Cd-substituted zinc finger protein, Sp1, retains its capacity to bind specifically to DNA [28].…”

Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

“…Studies of the reaction of apoMT and one of its component peptide sequences with Cd-carbonic anhydrase showed that both were remarkably effective in competing for protein-bound Cd 2+ [31,32]. There is evidence that, in vitro, Cd 2+ can substitute for Zn 2+ in zinc-finger structures and that such substitution can change the activities of the finger domains [22,28,33]. Therefore, this study has also examined how Cd-TFIIIA reacts with apo-or Zn 7 -MT.…”