From the Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202
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ABSTRACT |
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Preferential cleavage sites have been determined
for Fe2+/H2O2-mediated
oxidations of DNA. In 50 mM H2O2,
preferential cleavages occurred at the nucleoside 5' to each of the dG
moieties in the sequence RGGG, a sequence found in a majority of
telomere repeats. Within a plasmid containing a (TTAGGG)81
human telomere insert, 7-fold more strand breakage occurred in the
restriction fragment with the insert than in a similar-sized control
fragment. This result implies that telomeric DNA could protect coding
DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar
H2O2, preferential cleavage occurred at the
thymidine within the sequence RTGR, a sequence frequently found to be
required in promoters for normal responses of many procaryotic and
eucaryotic genes to iron or oxygen stress. Computer modeling of the
interaction of Fe2+ with RTGR in B-DNA suggests that due to
steric hindrance with the thymine methyl, Fe2+ associates
in a specific manner with the thymine flipped out from the base stack
so as to allow an octahedrally-oriented coordination of the
Fe2+ with the three purine N7 residues.
Fe2+-dependent changes in NMR spectra of duplex
oligonucleotides containing ATGA versus those containing
AUGA or A5mCGA were consistent with this model.
The cytotoxicity of hydrogen peroxide is mediated by highly
reactive oxidants generated by the reaction of reduced transition metals with H2O2 via Fenton
reactions (1).
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
DNA is a major target in H2O2-mediated
cell killing through such reactions (2, 3), but the oxidizing species
involved are probably not freely diffusible hydroxyl radicals
(·OH), but possibly a localized ·OH and/or related
iron-oxo species (4-8) whose properties are apparently governed by the
chelation state of the iron upon which they are generated (9). Because
DNA can chelate Fe2+ in several ways, DNA damage in
vivo in prokaryotes (2) and eukaryotes (10, 11), and in
vitro (2, 9) exhibit peculiar dose responses to
H2O2 concentration. The rates of killing or DNA
damage induction are maximal below about 1 mM, then drop
off and become independent of H2O2
concentration between approximately 5 and 50 mM. Based upon
these responses, we have defined two types of oxidants, termed types I
and II. Type I oxidants are scavenged by H2O2
to form less reactive species, exemplified by the reaction of
·OH with H2O2.
Hence, these oxidants appear to damage DNA only at lower
H2O2 concentrations; they are also scavenged by
alcohols. The constant rate of damage observed with
H2O2 concentrations between 5 and 50 mM was defined to be due to type II oxidants. In this case DNA damage might be induced by the reaction of
H2O2 with Fe2+ atoms that are
intimately bound to DNA such that nascent oxidants react with the DNA
before encountering exogenous scavengers such as
H2O2 or ethanol (9). Alternatively, one could
propose that type II oxidants do not react with
H2O2 or ethanol. In either case, these oxidants
would be formed on Fe2+ ions that are associated
differently within the DNA helix than those which give rise to type I oxidants.
Because of the high reactivity of ·OH and related oxidants,
damage to DNA in vivo or in the presence of scavengers
in vitro is thought to occur predominantly with such
radicals when they are generated by reaction of
H2O2 with metal ions bound to the DNA (12-14).
The binding of metals to preferred sequences within DNA and the
relationship of such associations to DNA damage by the Fenton reaction
have been the subject of recent investigations (13, 15-17), and it has
been proposed that transition metals in the presence of
H2O2 might cause sequence-specific damage to DNA (12, 18). However, whereas iron associated with DNA has been
repeatedly implicated in cytotoxic DNA damage (1, 3, 12, 14, 19, 20),
sequence preference for damage has been reported only with
copper/ascorbate (17) copper/NADH (21), with Fe2+ bound to
chelators or larger molecules (22), or with bleomycin (23). In fact,
Henner et al. (24) reported that the distribution of strand
breaks induced by excess Fe2+ (160 nM
DNA-nucleotide, 1 mM Fe2+, no added
H2O2) was indistinguishable from the
distribution upon treatment with -radiation and that strand breakage
was distributed equally (±20%) among the four nucleotides. However,
the DNA had been stored in distilled H2O and it is likely
that this DNA was not in a double-stranded, B-form.
In contrast, our laboratory found that nucleoside damage following
exposure of DNA to Fe2+ and H2O2
was not equally distributed among the nucleosides (25). Since we have
proposed that DNA damage in 0.5 mM
H2O2 was caused by Fe2+ ions that
associate differently with DNA than those which cause damage at 50 mM H2O2, we have now investigated
whether strand breaks vary in sequence location at the two
H2O2 concentrations. In this study, we
demonstrate that preferential cleavage sites do in fact differ for type
I (0.5 mM H2O2) and type II (50 mM H2O2) oxidants, and that these
sites can be grouped into small consensus sequences. These consensus
sequences, which appear to reflect specific modes of iron binding by
DNA, might be involved in regulatory processes of the cell.
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EXPERIMENTAL PROCEDURES |
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Materials-- H2O was deionized and then distilled. H2O2 was a 30% solution from Fisher Scientific; FeSO4·7H2O and NaCl (99.999%) were from Aldrich; ethanol (100%) was from Quantum Chemical Co. The highly purified NaCl was essential in order to avoid the accumulation of transition metals on DNA substrates. H2O2 and Fe2+ concentrations were measured as described previously (26). Reagents used for the Maxam-Gilbert sequencing were from Sigma and were freshly prepared according to Maxam and Gilbert (1977) (27). Restriction enzymes were from New England Biolabs (Beverly, MA); polynucleotide kinase was from Promega (Madison, WI).
CGCGATATGACACTAG, CGCGATAUGACACTAG, CTAGTGTCATATCGCG, CGCGATA5mCGACACTAG, and CTAGTGTCGTATCGCG were obtained from Operon Technologies (Alameda, CA) and were individually purified on a C18 HPLC1 column (10 mm × 25 cm, Econosphere; Alltech, Deerfield, IL) using a 5-15% acetonitrile gradient in 50 mM triethylammonium acetate, pH 7.3, and then repeatedly desiccated in the presence of ammonia to remove triethylamine. Each 16'-mer was then loaded onto the same column through which 1 M NaCl was pumped for 30 min followed by 30 min of H2O and then finally the sodium form DNA was eluted with 50% methanol (HPLC grade, Fisher). Duplexes were formed in 130 mM NaCl by the method of continuous fractions (28) and verified to be free of excess single-stranded oligonucleotide by NMR.
A plasmid containing human telomeric DNA, (TTAGGG)81, was a gift from Dr. Carol Greider, Cold Spring Harbor Laboratory. Plasmid pBluescript SK was from Stratagene, and plasmid pBSRep81 was constructed by inserting the telomere into the pBluescript SK. Escherichia coli XL1-Blue was used for plasmid maintenance and propagation. The plasmid DNA was purified by a Qiaprep Spin plasmid miniprep kit (Qiagen, Chatsworth, CA). The DNA was dialyzed at 4 °C against 1 mM EDTA, 50 mM NaCl for 24 h and then against 50 mM NaCl for 24 h.
Isolation of 144- and 191-bp PM2 DNA Fragment Substrates-- Bacteriophage PM2 DNA was isolated as described by Kuhnlein et al. (29), dialyzed against 0.1 mM sodium EDTA, 10 mM Tris-HCl, pH 7.5, for 2 days, and then extensively dialyzed against 130 mM NaCl. It was restricted with HindIII, and then the digest was separated on a 1.5% agarose gel into seven fragments of roughly 100, 250, 425, 450, 1000, 2100, and 5300 bp. A band containing the unresolved 425- and 450-bp fragments was cut out and the DNA electro-eluted into a dialysis bag with 0.089 M Tris borate, 2 mM EDTA, pH 7.8 (TBE). The eluate was extracted with 2-butanol (Aldrich), the volume was reduced to 0.5 ml, and then the DNA was precipitated three times with ethanol. The DNA pellet was dissolved in TBE and digested with HaeIII, and the digest was resolved on a 2.2% agarose gel.
With HaeIII, the 425-bp fragment generates 144- and 191-bp
fragments from the ends and a 90-bp fragment from the middle. (The 450-bp HindIII fragment generates 2 bands of about 68- and
382-bp.) To prepare 5'-33P-labeled 144- and 191-bp DNA
fragments, the mixture of the 425- and 450-bp HindIII
fragments was labeled with polynucleotide kinase and
[-33P]ATP before cleavage with HaeIII. The
end-labeled 144- and 191-bp fragments were then electrophoretically
separated on an 8% polyacrylamide gel. The gel slices were
individually electro-eluted into TBE buffer, and the eluted DNA was
extracted with phenol and chloroform and then precipitated with ethanol
and re-dissolved in 65 mM NaCl. Each fragment was then
dialyzed for 1 day each against 2 mM sodium EDTA, 5 mM Tris-HCl, pH 7.5, and 0.2 mM EDTA, 5 mM Tris-HCl, pH 7.5, and finally extensively against 65 mM NaCl (99.999%). The DNA was free of contamination by
transition metals as detected by the absence of nicking in the presence
of H2O2. To label the 3'-ends of the fragments,
the HindIII restriction fragments were treated with Klenow
DNA polymerase in the presence of 50 µM each dATP and
dGTP, and 40 µCi (0.5 µM) of
[
-33P]dCTP (2000 Ci/µmol, NEN Life Science
Products), and then the DNA was processed as described for the
5'-labeled fragments. The initial sequencing of the 144- and 191-bp
fragments was performed on both strands by the dideoxy procedure
according to the protocol supplied by United States Biochemicals.
DNA Damage Protocol for Determination of Strand Break
Locations--
A solution containing 2.5-8 µM
end-labeled restriction fragment in 65 mM NaCl, 20 nM to 2 µM FeSO4, 65 mM NaCl, and 10 mM ethanol as indicated, was
brought to a final concentration of either 0.5 or 50 mM
H2O2, and incubated for 25 min at room
temperature. A large excess of 0.3 M sodium acetate, pH
6.5, was then added, and the DNA was precipitated with ethanol. The
samples were centrifuged for 45 min at 23,000 × g, and
then the pellets were suspended and reprecipitated twice with ethanol
and finally dissolved in 7 µl of sequencing buffer (27). After
heating at 90 °C for 2 min, the resuspended damaged DNA was loaded
onto a pre-warmed 40-cm-long 8% polyacrylamide gel in 7 M
urea and electrophoresed at 60 watts for 3-4 h. Autoradiography was
performed by exposure of the dried gel to Kodak XAR-5 film for 3-10
days at 70 °C. Quantitation was accomplished by scanning the
autoradiogram and determining the area beneath a peak using ImageQuant
version 1.1 (Molecular Dynamics). For sequence markers, Maxam-Gilbert
sequencing reactions of undamaged substrate DNAs were loaded onto the
same gel. For detecting double strand DNA breaks after Fenton
reactions, the reacted DNA was analyzed on 8% polyacrylamide gels.
Protocol for Nicking of Plasmids-- The indicated amount of FeSO4 was added to 0.1 pmol of DNA in 130 mM NaCl and incubated at room temperature for the times indicated. Ethanol (100%) was then added where indicated to a final concentration of 10 mM (type I) or 100 mM (type II), and the oxidation was initiated by adding H2O2 to a final concentration of 50 µM (type I) or 50 mM (type II). The total volume was 10 µl. After 30 min at room temperature, EDTA was added to a final concentration of 50 µM and then forms I, II, and III of the plasmid DNA were separated by electrophoresis on 1% agarose gels. The gels were stained with ethidium bromide and also imaged, and the percentage of form I DNA quantified using an AlphaImager (Alpha Innotech, San Leandro, CA). Ethidium bromide stains form I one third less efficiently than forms II or III, and calculations were adjusted accordingly. The number of nicks per molecule was calculated from the amount of form I remaining, assuming a Poisson distribution.
Labeling and Restriction of Nicked Plasmid DNA--
Ten units of
T4 polynucleotide kinase and 2 pmol of [-33P]ATP were
incubated with 0.1 pmol of plasmid DNA in 50 mM Tris-HCl (pH 7.6) and 10 mM MgCl2. After 30 min at
37 °C, the kinase was inactivated by heating to 75 °C for 30 min
and the labeled plasmid DNA was digested with either 10 units of
HinfI in 50 mM NaCl, 10 mM Tris-HCl
(pH 7.9), 10 mM MgCl2, 1 mM
dithiothreitol or with 10 units of NciI in 50 mM
potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH 7.9) at 37 °C for 60 min. The
digests were separated by electrophoresis on 2% agarose gels. The gels were stained, imaged, and quantified as above, and subsequently dried,
and the bands were imaged and quantified using a PhosphorImager and
ImageQuant version 1.1.
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RESULTS |
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Strand Break Locations with Fe2+ and 50 mM H2O2 (Type II Oxidants)-- In order to study sequence preferences by types I and II radicals during iron-mediated Fenton reactions, we examined the location of cleavages after treating 144- and 191-base pair PM2 DNA restriction fragments with Fe2+ and either 0.5 or 50 mM H2O2 (Fig. 1).
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As predicted, cleavage sites differed for the two concentrations. In addition, the cleavages were predominantly within a few short specific sequences. When 50 mM H2O2 was present, preferential cleavage occurred at the nucleosides 5' to dG moieties in the consensus sequence, RGGG (Figs. 1 and 2, Table I), at some dC moieties in the sequence RCR, and at one dA moiety within the sequence YAAGA. (Bold underscored nucleotides indicate the sites of cleavage.) In the RGGG consensus sequence, the extent of cleavage was generally heaviest the more 5' the nucleotide resided in the sequence and the 3' dGMP was not preferentially cleaved. Three dC moieties were identified as preferential cleavage sites, two of which were in the consensus sequence, RCR (Table I, Figs. 1 and 2). Since 38 other dC residues in RCR were not preferentially cleaved under these conditions, this sequence by itself obviously does not constitute a preferential cleavage site. The extent of strand breakage in the presence of 50 mM H2O2 was not diminished by the presence of 100 mM ethanol, as would be expected for damage by a type II oxidant. When the DNA was exposed to Fe2+ and 50 mM H2O2 and then analyzed on polyacrylamide gels lacking urea, no evidence for the occurrence of double strand breaks was found (data not shown).
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Strand Break Locations with Fe2+ and 0.5 mM H2O2 (Type I Oxidants)-- With 0.5 mM H2O2, small amounts of cleavage occurred at RGGG and RCR sites, as would be expected if type II radicals were to form to a limited extent in 0.5 mM H2O2 (Table I, Figs. 1 and 2). However, preferential cutting in 0.5 mM H2O2 occurred mainly within the consensus sequences RTGR, TATTY, CTTR, and YAAGT. Also, as expected for cleavages by type I radicals, 10 mM ethanol inhibited all of these preferential cleavages (except those at RGGG) (Table I, Figs. 1 and 2). No double-strand breaks were observed. It is to be noted that controls (Fig. 2) include both the omission of H2O2 and, perhaps more significantly, the utilization of concentrations of Fe2+ that were too low to give rise to detectable breaks, even in the presence of H2O2. Five dC residues were cleaved in the RCR consensus sequence; however, 35 other dC residues in RCR were not preferentially cleaved under these conditions. Again, this sequence by itself obviously does not constitute a preferential cleavage site for type I oxidants.
The sequence RTGR was generally the most strongly cleaved. It appears 9 times in the two fragments and in all but one case, preferential cleavage was evident at the dT moiety (Table I; Fig. 1). (In the exception, 191-bp fragment, nucleotides 49-53, the RTGR is flanked by two cleavable TATTT sequences and opposite a cleaved ACG sequence.) The RTGR consensus sequence appears to be quite stringent, since only 2 of the 40 sequences in the two substrate DNAs that differ by one nucleotide (RT(A/Y)R, YTGR, or RTGY) and are not part of other cleavage sequences were preferential thymidine cleavage sites (Table I, Fig. 1).
All five TATTY sequences were preferential cleavage sites (Table I, Figs. 1 and 2). Breaks were also made at the two CATTR sites (base pairs 26 and 136, 191-bp fragment). These latter sequences may be grouped as non-consensus variants of either the RTGR or the TATTY consensus sequences. Alternately, these sites may constitute a separate consensus sequence.
The seven CTTR sequences present in the two fragments constitute preferential cleavage sites (Table I, Fig. 1 and 2C) for the 3' dTMP residues. Three additional preferential cleavage sites (base pairs 25 and 91 in the 144-bp fragment and base pair 80 in the 191-bp fragment) are non-consensus variants of this sequence.
Preferential cleavage at the 3' dAMP in the YAAGT was found in four of six of these sequences, albeit with less efficiency than with the aforementioned dTMP residues (Table I, Figs. 1 and 2). Two non-consensus variants, TAAGC (base pair 50, 144-bp fragment) and TGAGC (base pair 57, 144-bp fragment), may broaden the specificity of this consensus to RYAGY.
It is noteworthy that in only two cases (191-bp fragment, bp 98; 144-bp fragment, bp 60) was cleavage noted at a nucleotide that was 3' to a dCMP residue. Such a nick would have left a sugar fragment attached to dCMP, and such residues were noted by Bertoncini and Meneghini (11), to be uniquely absent from the termini of damaged DNA resulting from the treatment of CV1-P cells with H2O2. In addition, in no case were preferential cleavage sites immediately 5' to a dCMP, and in only 1 of 50 sites was there a pyrimidine nucleotide both 3' and 5' to the cleavage site (within the T8 run at bp 118 of the 144-bp fragment). Finally, all preferential cleavages at a purine nucleotide were 5' to another purine nucleotide. Cleavages are clearly far from random, and purine nucleotides strongly favor the presence of adjacent preferential cleavages, whereas cytosine nucleotides strongly discourage them.
Preferential Nicking of Telomeric DNA on a Plasmid by Type II Radicals-- Preferential cleavages by Fe2+/H2O2 at specific sequences could be due to preferential association of the iron at that sequence and/or due to electronic effects that attract and "trap" radicals formed elsewhere, as described by Hall et al. (30). The almost absolute specificity for nicking at RGGG in higher H2O2 concentrations is potentially significant, given that this sequence is characteristic of the large majority of telomeric repeats (31). (Exceptions among the telomeres of some fungi often have repeats containing RTGR.) Therefore, in order to determine whether telomeric DNA within a plasmid would sensitize the plasmid to type II strand breakage, we subjected an equimolar mixture of pBluescript plasmid and a pBluescript plasmid (pBSRep81) that contains an insert of a human telomere, (TTAGGG)81, to 50 mM H2O2 in the presence of Fe2+ and 10 mM ethanol. While the number of nicks per base pair increased with ferrous ion concentration, it remained equal between the two plasmids (Fig. 3). This equality held whether the plasmid was pre-incubated with iron for 5 or 120 min before H2O2 addition. Hence, while RGGG was identified as a preferential cleavage site, the telomere-containing plasmid did not sustain more nicks per base pair than the plasmid without the insert. However, at very high ferrous ion concentrations, considerably more double strand breakage was observed on the plasmid containing the telomere insert than on the plasmid without the insert (Fig. 3), suggesting that the single strand breaks may have been unequally distributed within the plasmid containing the telomere insert.
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To investigate the distribution of breaks within the
telomere-containing plasmid, we exposed the plasmid to
H2O2/Fe2+ and then radiolabeled the
strand breaks with T4 polynucleotide kinase and
[-33P]ATP. After inactivation of the kinase, the
plasmid was digested with HinfI. Among the restriction
fragments was a 842-bp fragment that contained the 480-bp telomere
insert and a total of 87 RGGG sequences (81 from the telomere and 6 from plasmid DNA). As a control fragment, we monitored a 1074-bp
fragment that contained 13 RGGG sites. Ethidium bromide staining and
quantitation confirmed complete digestion by the HinFI and
quantitated the DNA loaded and separated by agarose gel
electrophoresis. Autoradiography of the separated fragments allowed a
determination of the nicks put into each of the restriction fragments
(Fig. 4A).
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When the plasmid was pre-incubated for 5 min at room temperature with the Fe2+ prior to addition of H2O2, the fragment containing the telomeric DNA sustained 1.6-fold the number of nicks (Fig. 4A) of the control fragment. However, when the pre-incubation period was extended to 120 min, the telomeric DNA sustained 7-fold the number of nicks (Fig. 4B) of the control fragment. Increasing the pre-incubation period to 18 h did not further increase the ratio of nicks of the fragment with the telomeric DNA to that of the control fragment (data not shown). The ratio of RGGG sites containing the telomeric DNA to the control fragment is 6.7 (87:13), which corresponds to the aforementioned 7-fold increase in nicks. When the damaged and 33P-labeled DNA was digested by NciI, a similar ratio equal to the relative content of RGGG sites was observed, which depended upon pre-incubation of Fe2+ and DNA. In summary, it appears that Fe2+ ions are attracted to the two plasmids based upon their relative sizes. Then, in a time-dependent manner, these ions search out and bind to the RGGG sequences, which are targets for type II radicals.
These results support the observation that RGGG is the preferential cleavage site for type II oxidations and provides further evidence for the prediction that the iron-DNA associations that lead to type II oxidations are due to iron imbedded in the base stack next to purines (9). The requirement for pre-incubation of the Fe2+ and DNA would suggest a minimal contribution of the RGGG sequence acting as a sink of electrons (or electron holes) traversing the base stack, and favors specific Fe2+ binding.
Because the number of sensitive sites for type I radicals is large and includes sequences other than RGGG, the relative reactivity of the telomere-containing restriction fragment was investigated in 50 µM H2O2. The DNA and Fe2+ concentrations were kept the same as above, and the Fe2+ and DNA were pre-incubated for 120 min at room temperature before the addition of the 50 µM H2O2. The ratio of the labeling of the telomere-containing 842-bp fragment was 1.6-fold that of the control, 1074-bp fragment (Fig. 4C). This value is in agreement with the relative content of consensus sequences for type I radicals, RTGR, TATTY, CTTR, YAAGT, and RGGG, which is 101 versus 62, or a ratio of 1.63.
The Interaction of Fe2+ with the RTGR Consensus Sequence-- Among the preferential cleavages in micromolar H2O2 concentrations, cleavage at the RTGR thymidine was observed generally to be the strongest. In addition, as noted under "Discussion," RTGR appears to be a necessary promoter element for the regulation of certain genes which are responsive to oxidative or iron stress. Since the RTGR sequence has no apparent electronic distinctions, we explored ways in which Fe2+ might interact directly with the sequence ATGA in a duplex oligonucleotide, using the InsightII molecular modeling program (Fig. 5). Generally, the major groove is more distinctive than the minor groove for base distinction, particularly since the pyrimidines have C2 keto groups in the minor groove. However, attempts to place the Fe2+ between the N7 of the 5'-A of ATGA and that of the G in the major groove showed that the methyl group of thymine sterically interfered. Given the precedent of "base flipping" by the restriction methylases and repair DNA glycosylases (32, 33), we allowed the thymine to "flip out" of the base stack, possibly having been displaced by the Fe2+ or more likely to have spontaneously flipped out during "breathing" so as to allow the Fe2+ to enter. In this energy minimization modeling (Fig. 5A), the Fe2+ was allowed initially to coordinate the guanine N7. Coordination by the guanine O6 could then also occur via a water bridge (34), and Fe2+ is also attracted to the two phosphates that flank the thymidine. Steric hindrance between the Fe2+ and the thymine methyl in B DNA stabilizes the thymidine in the displaced position. The 5'-adenine then repositions such that its N7 takes part in the iron chelation. If this chelation is restricted to approximately 2 Å, surprisingly the 3'-adenine also repositions such that its N7 is within chelation distance of the iron. The three purine N7 residues and the phosphate between the dG and the dT would thus form a chelation pocket that is suitable for an octahedrally coordinated iron (Fig. 5B). If the guanine O6 also coordinates the iron via a water bridge, 5 of the 6 iron coordination sites are occupied. The last site, which is accessible to solvent, is juxtaposed to the thymidine deoxyribose which is the preferential cleavage site in the presence of 50 µM H2O2. Finally, the opposite strand remains essentially undistorted (Fig. 5, C and D).
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While these are predictions of a modeling exercise, a hypothetical scenario is clear. Fe2+ is attracted to the polyanionic DNA molecule and moves along the phosphate backbone among individual phosphate residues. It is attracted weakly by one or two purine N7 residues, but if in a RTGR sequence the dT moves (or had moved) aside and then the Fe2+ coordinates with the third purine N7, trapping it in a free-energy sink, at least long enough to show preferential cleavage at the RTGR sequence.
Using the energy minimization model as a working hypothesis, we monitored the effects of Fe2+ on the proton NMR signals of an ATGA-containing 16'-mer duplex. Iron was titrated into the DNA solution, and one-dimensional scans were taken (Fig. 6A). We noted iron-dependent changes in the chemical shifts of several imino region (12-14 ppm) and thymidine methyl region (0.5-2 ppm) peaks (Fig. 6, A and B). (Iron-dependent changes also were noted in the aromatic region notably in the T8 aromatic peak; data not shown.) Changes in the observed chemical shifts are centered around the ATGA sequence, and taper off toward the ends of the oligomer (Fig. 6B). To see if these changes were due to Fe2+ as opposed to Fe3+, we allowed the Fe2+ to oxidize. The observed changes in chemical shift reversed during a 16-h incubation under an aerobic atmosphere. Under nitrogen, however, the changes in chemical shift were stable (data not shown). Since the energy minimization model predicted that the thymine methyl in RTGR is a necessary trigger for this interaction, we tested this interaction with an oligonucleotide that contained an identical DNA sequence, except that the thymine in RTGR was replaced by a uracil (Fig. 6C). Clearly the broadening and changes in chemical shift seen for RTGR in Fig. 6 (A and B) did not occur in the presence of Fe2+. Hence, the thymine methyl plays a necessary role in the iron binding as predicted by the model.
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To further test which aspects of the thymine base might be necessary for interaction with Fe2+, we substituted the thymine in RTGR with a 5-methylcytosine (5mC) and on the opposing strand we introduced a corresponding guanine in lieu of the adenine. In this case a pattern of chemical shifts similar to that seen with the substrate containing RTGR was again evident (Fig. 6D). Notably, the 5mC methyl resonance was shifted downfield by 0.1 ppm. We can also compare the Fe2+-dependent change in chemical shift of the T8 methyl group to published differences in chemical shifts between stacked and extrahelical thymidine residues. Morden et al. (35) showed that the thymine methyl proton peak of a bulged extrahelical thymidine is 0.05-0.2 ppm downfield to the methyl proton peaks of all the paired and stacked thymidines. Moreover, Kalnik et al. (36) showed that when a bulged and unpaired thymine goes from a predominantly stacked to a predominantly extrahelical conformation, the methyl resonance of this thymine shifts downfield by up to 0.2 ppm. This latter unpaired thymine was part of an RTGR sequence (GTGA), so it is in an environment similar to the thymine of the ATGA sequence of our duplex oligonucleotide. In our case, the T8 (or the 5mC8) resonance was uniquely downfield shifted by ~0.1 ppm upon addition of Fe2+. Hence, the observed shifts in the presence of Fe2+ are consistent with the target pyrimidines becoming extrahelical as predicted by the simulation.
It must be noted that in Fig. 6A there was no
superimposition of spectra due to the presence of two species of DNA,
even though substoichiometric amounts of Fe2+ were used.
Moreover, we have noted that there is continuous shifting and
broadening of resonances upon increasing additions of iron (data not
shown). Therefore, it seems that compared with the chemical shift time
scale for NMR, the iron is in fast exchange among these small duplexes.
We had predicted that type I oxidants arise on Fe2+ ions,
which are loosely bound to DNA (26), and loose binding would give rise
to fast exchange. Alternately, the rapidity of exchange that we
observed might reflect the short length of the oligonucleotide, and
other physical means are called for to measure the stability of the
complex within longer, possibly circular DNAs.
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DISCUSSION |
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A definitive conclusion of this study is that different sequences
in duplex DNA undergo preferential nicking by Fe2+ in 0.5 mM versus 50 mM
H2O2. This result is consistent with the proposal that the different kinetics of DNA damage by Fe2+
and H2O2 at these two
H2O2 concentrations is attributable to radicals
formed on iron ions that interact differently with DNA and hence have
different properties. What was not anticipated, however, was the
adherence of preferential nicking to a small number of consensus
sequences which differed for the two H2O2 concentrations. It is unlikely that ·OH (or a similar oxidant)
could distinguish between different sequences of 4 bp. Thus we
presume that the preferential cleavages observed are at least partially
due to preferential iron binding at consensus sequences, although they
might also be due to an enhanced ability of Fe2+ to react
with H2O2 when so bound. Indeed,
electronegativity is not equally distributed along a DNA helix but is
dependent upon the local DNA sequence, and the binding of transition
metals to DNA is thought to involve association with phosphate residues followed by transfer to preferred sequence regions to form a complex whose stability depends upon the particular metal ion, the DNA sequence, and the secondary structure of the DNA in the region (37-41). It should be emphasized, however, that the location of DNA
nicks tells us where oxidants attacked the deoxyribose residues in DNA.
The next phase of these studies will be to look at which sequences
might give rise to particular base damages, e.g.
8-oxoguanine, thymine glycol, etc. Only after such studies will the
full spectrum of Fe2+ binding sites on DNA begin to emerge.
Many authors have pointed out that the strongest base component for chelation of transition metals on DNA is the purine N7 and that regions around a G-rich site are especially electronegative around the N7 atoms because of strong stacking interactions (39, 40, 42-44). The binding of iron to adjacent G residues would not be hindered by the exocyclic amino hydrogens of adenine and would possibly be supported by guanine C6 oxygens. The complex might also be stabilized by phosphate residues on the same strand which are 5' to the respective dG residues (42). Such an association with a guanine N7 and the 5'-proximal phosphate would place the Fe2+ in close proximity of the deoxyribose moiety 5' to the dG. This model correlates nicely with the observed cleavage pattern in GGG and with the decrease in ionization potential in the 3' to 5' direction (38, 44). Furthermore, GGG sequences might form sinks for oxidizing radicals (30, 44). The resistance of cleavages at RGGG sequences to ethanol and H2O2 might reflect the dispersion of a radical electron among the stacked guanines, thus shielding it from exogenous agents. It is noteworthy that copper/ascorbate/5 mM H2O2 (17) and Zn2+ (16) have recently been reported also to interact with DNA at runs of dGMP. In the future we plan to study the interaction of Fe2+ with the type II radical target, RGGG. These studies are especially interesting because of the presence of RGGG in a the large majority of telomere repeats. Should damage to DNA in vivo reflect what was observed with the plasmid studies, one might speculate that this consensus sequence in telomeres was selected to sequester transition metals which have been attracted to DNA in order to protect other sequences. However, this preferential binding could also contribute to H2O2-dependent telomere shortening and a reduction of the "Hayflick limit," which is observed in model systems of aging (45, 46).
In the RTGR consensus sequence, a Fe2+-DNA interaction in the minor groove would either place the iron too far from the guanine or too far from the preferentially cleaved sugar. On the other hand, if the iron were adjacent to the deoxyribose of thymidine in the major groove, the iron could also be within Van der Waals distance to the guanine N7. For sequences of more than two base pairs to interact with iron in the minor groove in the vicinity of the preferentially cleaved deoxyribose, the DNA must be distorted to the extent that it does not resemble B-DNA. On the other hand, with only small distortions of the B-structure of DNA, a Fe2+ ion in the major groove could access functional groups from 3 base pairs and still be in the vicinity of the preferentially cleaved deoxyribose moiety and chelating phosphates. Just as with protein-DNA interactions, it seems that metal-DNA sequence-specific interactions are more likely to occur in the major groove than in the minor groove (22).
As shown by NMR, the interaction of Fe2+ with the RTGR-containing 16'-mer duplex is readily reversible. This reversibility offers an explanation for the sensitivity of type I oxidations to high H2O2 concentrations. The Fe2+ might be oxidized by H2O2 while it is free, and the oxidant produced by this Fenton reaction quenched by H2O2 before damaging DNA. However, when formed within the RTGR sequence, the oxidant reacts immediately with DNA rather than with H2O2. One might then predict that Fe2+ interactions with RGGG would not be so readily reversible, as in this case damages are not quenched by H2O2 or ethanol.
The sequence RTGR occurs in several biologically interesting regions of
DNA. ATGG is part of the human and Drosophila centromeric repeats, AATGG, and the yeast repeat is AAATGG (47). Moreover, the RTGR
motif seems to occur as a required element in the regulatory regions of
genes that respond to iron- or oxidative stress. The consensus sequence
of the "iron box," a key promoter element involved in iron
homeostasis of E. coli, has two RTGR sequences in dyad symmetry (inverted repeats) (48). Two RTGR sequences in dyad symmetry
are also found in three consensus sequence elements in the promoter
that regulates the major mammalian AP endonuclease activity which is
necessary for the repair of the oxidatively damaged DNA (49). FRE1 is a
component of the principal yeast iron uptake system. The 5' non-coding
segment of FRE1 that is necessary for conferring iron-regulatory
transcriptional activity contains RTGR in repeated
GRTGAGCAAAAA sequences (50). It has been shown that the
RTGR motif is directly involved in the regulation of certain
chemoprotective responses mediated by the antioxidant-responsive element (ARE) (51), and it is interesting to note that
H2O2 induces gene activity under the control of
the ARE element. For example, the ARE that is found in the 5'-flanking
regions of the rat and mouse glutathione S-transferase Ya
subunit genes, the rat and human NAD(P)H:quinone reductase genes, and
-glutamyl cysteine synthetase genes each contains three RTGR
sequences. In deletion studies by Rushmore et al. (51), all
six of the deletions that infringe upon the RTGR motifs cause
significant reductions (>33%) in the basal and/or inducible promoter
activity. Only once in 13 other deletions was this response affected to the same degree (51). Further studies of the ARE consensus sequence showed that substitution of the dT in the core RTGR sequence resulted in complete loss of inducibility by chemoprotective inducers (52, 53).
The studies of Figs. 5 and 6 have implied a similar requirement for dT
in the specific interaction of RTGR with Fe2+, and a
similar interaction is not seen between RTGR and Fe3+.
Perhaps these regulatory regions are able to sense the oxidative environment of the cell via interactions of their RTGR motifs with
reduced iron.
We are currently determining the three-dimensional structure of the
interaction of Fe2+ with the small oligonucleotide complex
by two-dimensional NOESY. Once the structure is known, we plan to study
several of the naturally occurring regulatory motifs both physically
and biochemically in order to learn why this iron-binding sequence is
required in these regulatory motifs.
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ACKNOWLEDGEMENTS |
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We thank Matthew S. Falk for helping devise the PM2 DNA restriction scheme, and Corey Liu, Mark Kubinec, David Wemmer, and the University of California Berkeley College of Chemistry NMR facility for the use of the NMR machines and their assistance. The NMR machines are maintained by Instrumentation Grants DE FG05-86ER75281 from the U.S. Department of Energy and DMB 86-09305 and BBS 87-20134 from the National Science Foundation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R37GM19020, T32ES070075, and P30ES01896.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Sepracor, Inc., Marlborough, MA 01752.
§ Present address: Dept. of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143.
¶ Present address: Kosan Biosciences, Inc., Burlingame, CA 94010.
To whom correspondence should be addressed. Tel.:
510-642-7583; Fax: 510-643-5035; E-mail:
slinn{at}socrates.berkeley.edu.
The abbreviations used are: HPLC, high performance liquid chromatography; Y, pyrimidine; R, purine; N, any of the four DNA bases; bp, base pair(s); ARE, antioxidant-responsive element; TBE, Tris borate-EDTA; NOESY, nuclear Overhauser effect spectroscopy.
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REFERENCES |
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