(Received for publication, August 21, 1995; and in revised form, December 18, 1995)
From the
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
demonstrate the capability of iron to replace zinc in zinc finger
(hereby referred to as iron finger) both in vivo (using Escherichia coli BL21 (DE3)) and in vitro. Iron has
the ability to substitute for zinc in the ERDBD as demonstrated by
mobility shift and methylation interference assays of iron finger,
which show specific recognition of the estrogen response 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 ERDBD. More importantly, we demonstrate that iron finger in
the presence of HO
and ascorbate generates
highly reactive free radicals, causing a reproducible cleavage pattern
to the proximate DNA, the estrogen response element. The deoxyribose
method, used to detect free radical species generated, and the
resultant cleaved DNA ends, caused by iron finger, suggest that the
free radicals generated are hydroxyl radicals. Due to the close
proximity of the zinc finger to DNA, we postulate that iron-substituted
zinc finger may generate free radicals while bound to genetic
regulatory response elements, leading to adverse consequences such as
iron-induced toxicity and/or carcinogenesis.
The estrogen receptor (ER) ()is a nuclear hormone
receptor belonging to a superfamily of ligand-activated DNA-binding
transcription factors. Once ligand-activated, the ER regulates
transcription by binding to specific DNA response elements located
upstream from its target genes(1, 2) . Receptors of
the superfamily are divided into comparable, discrete domains known as
the ligand-binding, transcriptional activation, and DNA-binding
domains(3, 4) . The DNA-binding domain is the most
highly conserved region and is centrally located in the receptor.
Within the DNA-binding domain, eight highly conserved cysteines
tetrahedrally coordinate two zinc atoms, forming two nonequivalent zinc
finger-like motifs(5) . These two nonequivalent zinc fingers
fold to form a single structural module, enabling the receptor to bind
a specific hormone response element as a monomer or a
dimer(6) . In the case of the ER the estrogen response element
(ERE) is a 15-base pair DNA sequence consisting of two hexameric
palindromic sites separated by three base pairs,
AGGTCAXXXTGACCT(7) , facilitating a
``head-to-head'' homodimerization of two ERDBDs. As well as
binding the ERE, the ERDBD includes a nuclear localization signal (8) and a region mediating weak dimerization(9) .
Earlier in vitro studies have demonstrated the ability of cobalt and cadmium to structurally reconstitute the zinc finger motif of the ERDBD(10) . Nickel and copper on the other hand were shown to bind the ERDBD, yet neither restored the DNA binding property. In either case, the biological consequence of heavy metal incorporation into zinc finger may have relevance in the manifestation of metal-induced toxicity(10, 11) . Although metals such as iron, copper, or cobalt are essential elements, they are also toxic. Evidence suggests that elevated levels of iron contribute to an increased risk of cancer(12) . Primarily, this rise in the risk of malignancy is thought to be due to two reasons: the ability of iron to generate highly reactive free radicals and the increased demand for iron by the rapidly multiplying malignant cells needed for energy production (within the mitochondria) and DNA replication (ribonucleotide reductase)(13) . However, at this point, extensive studies investigating the mechanism of iron-induced toxicity have yet to define the true nature of this phenomenon.
This study
establishes the ability of iron to replace zinc in the ERDBD both in vivo (using Escherichia coli BL21 (DE3)) and in vitro. We report similar K values for DNA binding and metal:protein ratios for native
zinc finger and iron-replaced fingers. We also demonstrate the ability
of iron finger to generate free radicals that cause cleavage of the
proximate DNA, the ERE.
Our results demonstrate the ability of iron to replace zinc within the ERDBD. The gel mobility shift assay shown in Fig. 1demonstrates the ability of iron to replace zinc in the ERDBD in vitro. When apopolypeptide of ERDBD was dialyzed against buffer containing iron as Fe(II) the specific DNA binding activity was restored to about the same level of the native, zinc finger (Fig. 1, lanes a and c, respectively), while the control, apopolypeptide dialyzed against metal-free buffer, showed no binding activity (Fig. 1, lane d).
Figure 1: Mobility shift experiments with native, apopolypeptide, iron in vivo and in vitro replaced ERDBD, and the ERE. Lane a, iron in vitro substituted finger; lane b, apopolypeptide; lane c, native ERDBD zinc finger; lane d, apopolypeptide control; lane e, in vivo iron finger; lane f, free DNA. Experimental details are described under ``Materials and Methods.''
The iron finger of ERDBD could be synthesized in vivo when the transformed E. coli were grown in minimal medium supplemented with 50 µM ferrous ammonium sulfate . As seen in Fig. 2, transformed cells displayed similar growth rates when grown in either a metal-depleted or a zinc-supplemented medium, while the cells clearly showed a much faster growth rate in the iron-supplemented medium. This result is in accord with the assumption that a continuous supply of iron is essential for the growth and reproduction of these organisms(13) . The ERDBD purified from E. coli culture grown in the iron-supplemented minimal medium had similar DNA binding specificity to ERE as that of native ERDBD, and the iron finger formed in vitro (See Fig. 1, lane e). Metal analysis of purified in vivo iron finger revealed a 2:1 molar ratio of iron to ERDBD peptide (2.3 ± 0.2 atoms of Fe/mol of peptide), confirming that the peptide was synthesized as iron finger. Zinc content of this peptide was only 0.02 ± 0.01 atoms/mol of ERDBD.
Figure 2: Growth rate curves of transformed E. coli BL21 (DE3). The bacteria were grown in minimal media 9 supplemented with 50 µM iron or zinc or no metal (control). See details under ``Materials and Methods.''
The methylation
interference assays with the half-site ERE oligonucleotide, performed
with zinc- or iron-ERDBD, each displayed specific interactions only
with the guanine of the ERE half-site hexamer (TGACCT) (Fig. 3)
as previously reported(10) . The above results, combined with
the comparable ERE-ERDBD K values obtained for
native ERDBD and in vivo and in vitro iron fingers (K
values of 10, 29, and 39 nM,
respectively) evidently show that iron can substitute for zinc in the
ERDBD and maintain ERE-specific binding and affinity. The zinc content
of iron finger formed in vivo is too low to account for the
above results, and in vitro formed iron finger had no
detectable zinc present yet showed identical results to in vivo iron finger.
Figure 3: Methylation interference assays of native and iron in vivo and in vitro replaced ERDBD with ERE hexamer containing oligonucleotide complexes. Lane f, free DNA; lane a, DNA bound by native zinc finger; lane b, DNA bound by in vitro iron finger; lane d, DNA bound by in vivo iron finger. The ERE half-site sequence is indicated in boldface type. See details under ``Materials and Methods.''
It is known that iron induces mutagenesis/carcinogenesis(22, 23, 24) , but the exact mechanism of iron-induced toxicity is unknown. An excess of iron in the circulatory plasma, which exceeds the iron saturation capacity of serum, results in abnormal deposition of iron in body organs, which may lead to deleterious effects. Stevens et al.(12) found that levels of transferrin saturation between 50 and 60% increase the risk of cancer by 37%. The ability of iron to promote malignancy is believed to be a result of at least two possible mechanisms. One is the catalytic interaction of iron and oxygen, generating free radicals and resulting in cellular consequences such as DNA mutations, sister chromatid exchange, and carcinogenesis. This mechanism is supported by the observations that cells are protected against iron-induced toxicity by either sequestering iron with iron chelators or eliminating hydrogen peroxide with catalase and thereby preventing the induction of free radicals(25, 26) . Second, iron acts as an essential element for the growth and replication of neoplastic cells(27) . Iron is an essential element for key enzymes in DNA synthesis and in the respiratory chain(28) . Moreover, when cellular iron levels are elevated, both mechanisms may act in synergy. Iron-generated free radicals could lead to cellular transformations forming neoplastic cells, and following such an event, the high availability of this same iron could aid in the growth and proliferation of these cancer cells.
The
hydroxyl radical can travel a maximum distance of 60
Å(25) . Therefore, the resultant cellular damage occurs
proximate to the site of formation. Hydroxyl radicals produced near DNA
may cause strand breakage, while damage done to protein that is present
in excess may have no biological consequences. Therefore, the
biological importance of hydroxyl radical formation is related to the
site of action. To investigate the damage done by the iron finger to
the ERE, a P-5`-labeled 37-base pair oligonucleotide,
containing the ERE, was incubated with iron finger in the presence of
0.03% H
O
and 1.0 mM ascorbate in 10
mM Tris-HCl buffer, pH 7.4, and run on a 20% urea denaturing
gel. Free iron and Fe-EDTA cleaves both DNA strands randomly (Fig. 4, lanes c-f), while we find a reproducible
specific cleavage pattern (Fig. 4, lanes i and j) when iron finger is incubated with the ERE. In Fig. 4, the bands caused by free iron, Fe-EDTA, and iron finger
scission are aligned. These results suggest that the mechanism
of scission and, therefore, the species responsible are the same in
each case, involving specifically the hydroxyl radical(29) .
This is consistent with the results obtained from the deoxyribose
method. This raises the question: are the observed iron finger-ERE
scissions caused by iron finger or a result of a footprint, such as one
that would be obtained by incubating free iron with a protein-DNA
complex? Here again we have a
P-5`-labeled 37-base pair
oligonucleotide containing the ERE, used for a zinc finger DNase
footprint or incubated with iron finger or zinc finger in the presence
of 0.03% H
O
and 1.0 mM ascorbate in 20
mM phosphate or 10 mM Tris-HCl, pH 7.4, with or
without 10 µM Fe(II), and the cleavage products were
analyzed on a 20% urea denaturing gel. As seen in Fig. 5(lane b), the DNase footprint of native finger
has no scissions occurring within the ERE site, in contrast to iron
finger cleavage (Fig. 5, lane c). Moreover, zinc finger
incubated with free Fe(II) (10 µM) (Fig. 5, lane d) has a cleavage pattern distinct from that of iron
finger. DNA labeled at the 5`-end incubated with free Fe(II) and under
the appropriate conditions creates DNA fragments that terminate in a
3`-phosphate and 3`-phosphoglycolate groups, and chelated iron, such as
Fe-EDTA, leaves fragments ending in only 3`-phosphate
groups(29) . The same cleavage experiments performed with
phosphate buffer instead of Tris buffer with 10 µM free
iron gives the double scissions per base (Fig. 5, lane
f), while iron finger or Fe-EDTA (Fig. 5, lanes c and g) do not. Therefore, we conclude that the DNA strand
breaks are mediated through iron finger generating free radicals while
coordinated to the ERDBD and not from the oxidative release of iron
finger iron.
Figure 4:
Cleavage of ERE containing DNA by iron
finger. Each strand of ERE (I and II) was separately 5`-labeled and
incubated with free iron, Fe-EDTA, and iron finger in the presence of
HO
and ascorbate. Lane a, ERE(I)
alone; lane b, ERE(II) alone; lane c, ferrous
ammonium sulfate and ERE(I); lane d, ferrous ammonium sulfate
and ERE(II); lane e, Fe-EDTA and ERE(I); lane f,
Fe-EDTA and ERE(II); lanes g and h, G-methylation
Maxam-Gilbert sequencing of ERE(I) and ERE(II), respectively; lane
i, iron finger and ERE(I); lane j, iron finger and
ERE(II). The ERE sequence is indicated in boldface type. See
details under ``Materials and
Methods.''
Figure 5:
DNase and hydroxyl radical footprint of
ERE containing DNA. ERE containing DNA 5`-labeled on one strand (ERE I) was treated with DNase in the absence or presence of
native or iron finger (lanes a-c, respectively).
5`-labeled ERE(I) incubated with free iron and/or zinc finger, Fe-EDTA,
and iron finger in the presence of HO
and
ascorbate in phosphate or Tris-HCl is shown in lanes
d-h. Lane d, native finger and free iron; lane
e, native alone; lane f, ERE(I) and free iron in
phosphate buffer; lane g, ERE(I) and Fe-EDTA; lane h,
G-methylation Maxam-Gilbert sequencing of ERE(I). See details under
``Materials and Methods.''
The specificity of iron-ERDBD binding and cleavage at the specific DNA binding target versus other DNA sequences were evaluated. Iron-ERDBD protein demonstrates specific binding and cleavage to the target DNA only (data not shown).
The ERDBD binds to the ERE as a ``head-to-head'' dimer. Therefore, when iron-substituted ERDBD binds the ERE there are four individual iron fingers proximate to the DNA. To investigate in detail the specific damage done by iron finger to the ERE, the resultant cleavage pattern is aligned to a schematic of the dimerized ERDBD docked on to the ERE using the crystal structure coordinates (Fig. 6). The SETOR program (30) was used to produce the molecular diagram of the ERDBD bound to DNA. The cleavage pattern was sequenced and aligned using G-methylation cleavage with piperidine and DNA size markers. Below the cleavage pattern a histogram demonstrates the relative damage done to the ERE. The data points were calculated using NIH Image 1.52 computer software. These results demonstrate that possibly all four iron fingers are involved, to some extent, in cleaving the ERE. The ERDBD has two nonequivalent zinc fingers, each with a distinct function. One finger mediates the specific binding of the ER to the ERE by binding within the major groove of the DNA. It is this iron-substituted finger, binding within the major groove of the ERE and therefore proximate to the DNA, that causes the most extensive damage to the DNA. Furthermore, as seen from the histogram of Fig. 6, the damage done by the iron finger, within the major groove, has a gaussian distribution, demonstrating that metal-induced DNA cleavage is not site-specific. The second ER finger includes a dimerization face responsible for the ER head-to-head dimer interface. The dimerization finger hovers above the ERE and hence is further away from the DNA, yet it still cleaves the ERE, but to a lesser extent.
Figure 6:
The specific damage done by iron finger to
the ERE. The resultant cleavage pattern is aligned to a schematic of
the dimerized ERDBD bound to the ERE using the crystal structure
coordinates. Below the cleavage pattern a histogram demonstrates the relative damage done to the ERE. The asterisk designates the P-5`-end-labeled-strand of the double
helix.
In the case of an excess of iron, the metal may be incorporated into the zinc fingers directly during its synthesis and folding or by means of a zinc/iron exchange. Zinc, in zinc finger, appears to be kinetically labile and is exchangeable(31, 32) . NMR studies have revealed that a chemical exchange process occurs in the zinc binding site(33) . Iron finger, along with hydrogen peroxide, produced from cellular processes, and a reducing agent, such as ascorbate or superoxide, will drive the Fenton/Haber and Weiss reactions forward, augmenting the rate of hydroxyl radical generation. The hydroxyl radicals generated, that escape through the iron finger peptide, may cause extensive damage to the proximate DNA, the ERE. Therefore, we postulate that an iron-substituted zinc finger may generate free radicals while bound to the ERE, leading to adverse consequences such as iron-induced toxicity and/or carcinogenesis.