From the Sealy Center for Molecular Science and the Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1071
Received for publication, November 25, 2002
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ABSTRACT |
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DNA-protein cross-links (DPCs) are formed upon
exposure to a variety of chemical and physical agents and pose a threat
to genomic integrity. In particular, acrolein and related aldehydes produce DPCs, although the chemical linkages for such cross-links have
not been identified. Here, we report that oligodeoxynucleotides containing 1,N2-deoxyguanosine adducts of
acrolein, crotonaldehyde, and trans-4-hydroxynonenal can
form cross-links with the tetrapeptide Lys-Trp-Lys-Lys. We concluded
that complex formation is mediated by a Schiff base linkage because
DNA-peptide complexes were covalently trapped following reduction with
sodium cyanoborohydride, and pre-reduction of adducted DNAs inhibited
complex formation. A previous NMR study demonstrated that duplex
DNA catalyzes ring opening for the acrolein-derived DNA-protein cross-links
(DPCs)1 are produced upon
exposure to several exogenous and endogenous agents, including ionizing
radiation, metal compounds, oxygen radicals, X-rays, and reactive
aldehydes (1-6). The histones and nuclear matrix proteins are the
predominant substrates involved in DPC formation (7-9), and chromatin
structure significantly affects cross-linking efficiency (10-12). Not
surprisingly, aldehydes with established DPC-forming ability disrupt
DNA replication for the SV40 minichromosome following exogenous
exposure (13), suggesting that DPC damage presents a major obstacle to
the mammalian DNA replication (and transcription) machinery. We
envisage that a DNA repair and/or damage avoidance pathway exists to
prevent interruptions to these normal cellular events, although a
unified repair scheme has not been elucidated for all DPC lesions. In particular, studies conducted in xeroderma pigmentosum cells have implicated nucleotide excision repair (NER) in the removal of DPCs
induced by trans-Pt(II)diammine dichloride (1); however, studies on formaldehyde-induced DPCs indicate that NER is a dispensable pathway in the active repair of these lesions (14-16).
Among the agents that induce DPCs, acrolein and crotonaldehyde are
bifunctional electrophiles belonging to a group of highly reactive
aldehydes termed 2-alkenals. These compounds retain two electrophilic
reaction centers and are capable of forming various DNA and protein
adducts as well as DPCs (2, 17-19). It has been postulated that the
2-alkenals and also the structurally related 4-hydroxy-2-alkenals
(e.g. trans-4-hydroxynonenal (HNE)) represent significant sources of endogenous DNA damage because of their presence
as metabolites of lipid peroxidation (19, 20). Acrolein and
crotonaldehyde are known carcinogens and pose an environmental health
risk as constituents of automotive exhaust and tobacco smoke (21, 22);
however, because these 2-alkenals cause damage to a multitude of
cellular macromolecules, what role DPC formation plays in their
observed mutagenic and carcinogenic effects is as yet unclear.
Likewise, although the formation of 4-hydroxynonenal-derived protein
adducts has been correlated with degenerative conditions such as
cardiovascular and Parkinson's diseases (23, 24), demonstration that
HNE can induce DPCs may suggest alternative mechanisms to explain the
observed cytotoxicity of this compound.
In the case of formaldehyde- and malondialdehyde-induced DPCs, the
sequence of reactivity in cross-link formation appears to involve a
rapid primary reaction to form a protein adduct, followed by a slower
secondary reaction with DNA amines to form a DPC (25, 26). However, the
detection of stable acrolein-, crotonaldehyde-, and
4-hydroxynonenal-derived DNA adducts in vivo (27-29)
suggests that bifunctional electrophiles can react to form primary DNA
adducts capable of participating in secondary reactions with proteins.
Acrolein reacts with DNA to form a major exocyclic adduct,
Materials--
All peptides were prepared by the NIEHS
Center in Environmental Toxicology-Protein Chemistry Laboratory
(University of Texas Medical Branch). Sodium borohydride was obtained
from Sigma, and sodium cyanoborohydride was obtained from Aldrich.
[ Peptide Quantitation--
Following initial syntheses, peptides
were analyzed by mass spectrometry, and the composition of each was
confirmed by observing a major peak corresponding to the predicted
molecular mass. Peptides were subsequently purified by preparative HPLC
and resuspended in a solution of 20:80 acetonitrile/water. Because all
peptides used in this study contained a single Trp residue, the
concentration of each peptide solution was determined by monitoring Trp
absorbance at 280 nm using a Shimadzu BioSpec-1601 spectrophotometer.
Concentrations were calculated using 5500 M Adducted Oligodeoxynucleotides--
The synthesis of adducted
deoxynucleosides was carried out by Nechev et al. as
described previously for the proximal deoxyguanosine adduct of acrolein
(32) and the (6R,8R)- and
(6S,8S)-crotonaldehyde adducts (33). These
adducted deoxynucleosides were constructed into 12-mer
oligodeoxynucleotides with the sequence 5'-GCTAGCG*AGTCC-3', where G* denotes the adducted base. Oligodeoxynucleotides containing the acrolein and crotonaldehyde adducts were a generous gift
of Drs. Constance Harris and Thomas Harris (Vanderbilt University). The
stereospecific synthesis of the HNE-adducted deoxynucleosides has been
described previously (34). The synthesis of oligodeoxynucleotides containing HNE-derived adducts was carried out by Hao Wang (Vanderbilt University); these DNAs were a generous gift of Dr. Carmelo Rizzo (Vanderbilt University). Following syntheses, all adducted
oligodeoxynucleotides were purified by HPLC and
Determination of Duplex DNA Integrity--
The complement to the
adducted 12-mers, with sequence 3'-CGATCGCTCAGG-5', was synthesized by
Midland Certified Reagent Co. and was gel-purified by standard
procedures prior to use. For the preparation of duplex DNAs, adducted
12-mers were annealed to the complementary strand (20-fold molar
excess) in 1 M NaCl by heating at 90 °C for 3 min and
cooling slowly to 4 °C. For experiments requiring single-stranded
DNA in trapping reactions, the annealing step was omitted. To verify
the integrity of the double-stranded substrate for trapping reactions,
single- and double-stranded DNAs were incubated under standard reaction
conditions (50 mM HEPES (pH 7.0) and 100 mM
NaCl) for 30 min at 4 °C. To each reaction were added 0.2 volumes of
loading buffer (0.25% (w/v) bromphenol blue, 0.25% xylene cyanol, and
40% (w/v) sucrose in H2O), and DNAs were analyzed on a
12.5% native polyacrylamide gel in 0.5× buffer containing 45 mM Tris borate and 1.0 mM EDTA. Duplex DNAs
were visualized as bands migrating with slower mobility relative to the
labeled single-stranded oligodeoxynucleotides, and quantitative
annealing was observed for all duplexes (data not shown).
Trapping of Covalent DNA-Peptide Complexes Using
NaCNBH3--
For standard trapping reactions, adducted DNA
(75 nM) was incubated with peptide in 50 mM
HEPES (pH 7.0) and 100 mM NaCl at 4 °C. An aqueous
solution of NaCNBH3 was prepared on the day of use and
added to each reaction (final concentration of 50 mM) immediately preceding the addition of peptide. Unless otherwise stated,
the concentration of peptide in the trapping reactions was 1.0 mM, and reactions were quenched by the addition of a
freshly prepared aqueous solution of NaBH4 (final
concentration of 100 mM). Each reaction mixture was
subsequently diluted 5-fold by the addition of 1.25× loading buffer
(59% (v/v) formamide, 12.5 mM EDTA, 0.012%
(w/v) bromphenol blue, and 0.012% xylene cyanol) and heated at
90 °C for 3 min. An aliquot of each reaction was loaded onto a 15%
denaturing polyacrylamide gel (8.3 M urea) in sequencing
buffer (134 mM Tris base, 44 mM boric acid, and
10 mM EDTA), and products were separated by electrophoresis
for 5 h at 1500 V.
Illustrations--
Results were visualized from wet gels by
PhosphorImager analysis, and product bands were quantitated using
ImageQuant Version 5.0 software. In all cases, the trapped Schiff base
complex was calculated as the amount of major shifted complex on the
gel as a percentage of the substrate DNA + major shifted complex.
Electronic gel files were processed with Adobe Photoshop Version 5.5 and Illustrator Version 9.0.
Covalent Trapping of Lys-Trp-Lys-Lys at the Peptide Trapping Kinetics for Aldehyde-derived
1,N2-Deoxyguanosine Adducts--
We next investigated the
possibility that other 1,N2-deoxyguanosine
adducts, i.e. crotonaldehyde- and HNE-derived adducts (Fig. 1), could undergo ring opening in duplex DNA in a fashion analogous to
the Inhibition of Cross-link Formation by Pre-reduction of
Aldehyde-adducted DNAs--
To provide further evidence that the
observed complexes were mediated by Schiff base chemistry, adducted
oligodeoxynucleotides were pretreated with the strong reducing agent
NaBH4 to inhibit DNA-peptide cross-link formation. As
stated above, NaBH4 reduces an aldehydic function to a
primary alcohol, thereby rendering this group nonreactive with
amines; thus, if the observed DNA-peptide complex in the trapping assay
was the result of a Schiff base linkage, we reasoned that pre-reduction
of the adducts should prevent cross-link formation. A previous
investigation demonstrated that the half-life of a nonreduced abasic
site aldehyde is 12 s at pH 6.8 in the presence of 100 mM NaBH4 (37); however, we could not
confidently predict that the reduction kinetics of the adducts under
study here would be equally fast. Specifically, to allow the
opportunity for the subpopulation of the ring-closed conformer of each
adduct to shift toward a readily reducible ring-open form, substrates
were preincubated with 75 mM NaBH4 for 1 h
to achieve quantitative reduction of the ring-open aldehyde adducts. For NaBH4 trapping inhibition analysis, each of the
1,N2-deoxyguanosine-adducted
oligodeoxynucleotides that reacted with high relative efficiency in our
trapping assay was selected, viz. the acrolein-derived
Reactivity of Single- Versus Double-stranded Aldehyde-adducted
DNAs--
As stated above, a prior NMR study of the Investigation of the Reactive
Nucleophile--
Peptides capable of catalyzing It has been well documented that aldehydes react with
biomolecules to form a variety of adducts, many of which have been
correlated with mutagenic, carcinogenic, and cytotoxic consequences. In
particular, acrolein and crotonaldehyde were shown previously to form
protein adducts at lysine residues by Schiff base and Michael addition pathways (17, 18); however, although these compounds can also produce
DPCs, the chemistry underlying the formation of these adducts has not
been elucidated. We are unaware of any prior study demonstrating that
HNE can produce DPCs, although Uchida and Stadtman (40) proposed that
HNE-derived Michael addition products may participate in secondary
reactions to form lysine-mediated inter- and intrasubunit protein
cross-links. This observation suggests that an analogous reaction of
HNE with a protein lysine and a nucleic acid amine might represent a
plausible pathway in the formation of DPCs. In addition, the detection
of 1,N2-deoxyguanosine adducts of acrolein,
crotonaldehyde, and 4-hydroxynonenal in human and rodent tissues, both
as endogenous adducts and following chemical treatment (27-29,
41-43), may indicate that these stable DNA adducts exist as
intermediates along a pathway of formation for DPCs. Consistent with
such a mechanism, the results presented here demonstrate that peptide
amines can react with these DNA adducts to form DNA-peptide cross-links
via Schiff base linkage. We propose that this pathway may account for a
subset of DPCs formed within the cell whereby nucleophilic functions on
proteins react with primary aldehyde-derived DNA adducts. For example, the case in which a lysine-rich histone protein is juxtaposed with a
1,N2-deoxyguanosine adduct, as might be found in
the nucleosome, may provide the proper scenario for such a reaction. As
an alternative to this mechanism, Voitkun and Zhitkovich (26)
demonstrated previously that malondialdehyde (a bifunctional
electrophile and known DPC inducer) reacts preferentially with the
histone H1 protein compared with DNA in vitro, leading to an
adducted protein intermediate that precedes the formation of a DPC.
However, a number of investigations have established that the
malondialdehyde-derived
3-(2'-deoxy- The borohydride trapping methodology employed in this study
was utilized previously to isolate Schiff base intermediates in peptide-catalyzed A major factor affecting the formation and stability of a Schiff base
complex should be the pKa of the reactive amine because nucleophilic attack at the ring-open aldehyde adducts requires
a neutral deprotonated amine. Here, we found that the preferred
reactive nucleophile for peptides is located at the As depicted in Fig. 2B (species 1), a
requisite step in the reaction of peptides to form DNA-peptide
cross-links with the DNA adducts under study here is the ring opening
of the adduct to form a reactive aldehyde. Recently, an NMR study
confirmed that duplex DNA catalyzes rearrangement of the ring-closed
Although a significant amount of literature has enumerated the various
agents inducing DPC damage, the potential for repair has not been
clearly elucidated for these DNA lesions. When the 16-kDa DNA repair
enzyme T4 pyrimidine dimer glycosylase is covalently attached to
DNA as an artificial DPC lesion, the bacterial UvrABC NER proteins are
able to recognize this damage and to initiate repair in
vitro at a rate comparable to that observed for other well
characterized NER substrates (53). In mammalian cells, NER has
been implicated in the repair of DPC lesions for only a select number
of DPC-inducing agents, possibly suggesting that recombinational
pathways participate in the cellular response to DPCs.
Furthermore, the active removal of formaldehyde-induced DPCs in
human cells is inhibited upon treatment with lactacystin, a specific
proteasome inhibitor (16). Thus, an attractive model for the repair of
DPC lesions may include the targeting for proteolysis of proteins that
have become covalently attached to DNA (16). Although it remains to be
confirmed that Schiff base-mediated cross-links represent biologically
significant chemical linkages, the ability to generate site-specific
DNA-peptide cross-links now provides a tool to evaluate the repair
model described above. Specifically, the cross-links generated in this
study may represent mimics of biologically relevant DNA-peptide
complexes that may exist as intermediates along a pathway of repair for
DPCs. Experiments are currently underway to test this model.
-hydroxy-1,N2-propanodeoxyguanosine
adduct to yield an aldehydic function (de los Santos, C., Zaliznyak,
T., and Johnson, F. (2001) J. Biol. Chem. 276, 9077-9082).
Consistent with this earlier observation, the adducts under
investigation were more reactive in duplex DNA than in single-stranded
DNA, and we concluded that the ring-open aldehydic moiety is the
induced tautomer in duplex DNA for adducts exhibiting high relative
reactivity. Adducted DNA cross-linked to Arg-Trp-Arg-Arg and
Lys-Trp-Lys-Lys with comparable efficiency, and
N
-acetylation of peptides dramatically
inhibited trapping; thus, the reactive nucleophile is located at the
N-terminal
-amine of the peptide. These data suggest that Schiff
base chemistry can mediate DPC formation in vivo following
the formation of stable aldehyde-derived DNA adducts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxy-1,N2-propanodeoxyguanosine
(
-HOPdG); and recently, this adduct was shown to undergo ring
opening in duplex DNA to yield an aldehydic moiety (30). Consistent
with the predicted reactivity of the ring-open aldehydic tautomer, the
-HOPdG adduct was shown to form an interstrand DNA-DNA cross-link to
the N2-position of an opposing guanine base in a
5'-CpG sequence context, mediated by a Schiff base (or carbinolamine)
linkage (31). To test the possibility that peptide amines might also
provide suitable substrates for cross-link formation, we evaluated the
propensity for the major 1,N2-deoxyguanosine
adducts of acrolein, crotonaldehyde, and HNE (Fig. 1) to form cross-links with the
tetrapeptide Lys-Trp-Lys-Lys. These DNA adducts were synthesized
previously for acrolein (32), crotonaldehyde (33), and HNE (34), and
each was constructed into a 12-mer oligodeoxynucleotide for use
in this study. We utilized a borohydride trapping methodology to
covalently trap Schiff base-mediated DNA-peptide complexes at the
ring-open aldehydic adducts, similar to methodology used recently in
probing the catalytic mechanism of peptide-catalyzed
-elimination at
abasic sites (35). Additionally, the reactivity of each adduct
was assessed in single-stranded and duplex DNAs, and a characteristic
amine capable of forming such a cross-link was identified.
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Fig. 1.
Structures of the
1,N2-deoxyguanosine adducts of acrolein
(a), crotonaldehyde (b and
c), and HNE (d-g).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was obtained from PerkinElmer Life Sciences.
1
cm
1 as the Trp molar extinction coefficient (36).
-32P-labeled on the 5'-end with T4 polynucleotide kinase
following standard procedures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HOPdG
Adduct--
A prior NMR study on the
-HOPdG adduct showed that this
exocyclic ring-closed adduct is able to tautomerize to a ring-open aldehydic moiety in duplex DNA (Fig.
2B, species 1 and
2), a result that may be rationalized, at least in part,
because of stabilization of the ring-open structure by pairing of a
cytosine base opposite the lesion (30). This structural observation
prompted our investigation into the reactivity of the
-HOPdG adduct
with peptides and, in particular, the ability of this adduct to form a
Schiff base-mediated cross-link between a peptide amine and the
aldehydic function at the adducted deoxyguanosine (Fig. 2B,
species 3). In the presence of a reducing agent, such a
complex can be trapped as a reduced Schiff base (Fig. 2B,
species 4); thus, formation of this stable covalent species
was monitored as the experimental end point in cross-link formation.
For the trapping assay, a 12-mer oligodeoxynucleotide substrate
containing a centrally located
-HOPdG adduct (Fig. 2A)
was obtained, and this DNA was reacted with the Lys-Trp-Lys-Lys peptide
in the presence of NaCNBH3. In this experiment, a complex was observed that migrated slower on a denaturing polyacrylamide gel
compared with the adducted 12-mer, and the amount of gel-shifted complex that was observed was dependent on the concentration of peptide
in the reaction (Fig. 2C). A shifted DNA band that migrated only slightly slower than the substrate DNA was also observed when
reducing agent was present without peptide; this band was competed away
by the addition of increasing peptide concentrations. One possibility
may be that this species represents a subpopulation of substrate DNA
(with altered gel mobility) in which the ring-open
-HOPdG aldehyde
has reacted to form a Schiff base complex with Tris base in the
reaction mixture. Such a complex should also be reducible to form a
stable covalent species, and experiments are currently underway to test
this hypothesis utilizing mass spectrometry to identify the chemical
composition of this species.2
Very low levels of complex were observed in the absence of reducing agent for the
-HOPdG-adducted DNA; and when non-adducted DNA was
examined as a negative control, a shifted complex was not observed in
the presence or absence of NaCNBH3.
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Fig. 2.
A, shown is the sequence of the 12-bp
oligonucleotide used in this study. The position of the adducted
deoxyguanosine is denoted by an asterisk on the upper stand,
and the position of the 32P label is shown. B,
shown are the structures of the ring-open (species 1) and
ring-closed (species 2) forms of the acrolein-derived
-HOPdG adduct. A peptide amine reacts with the aldehydic DNA adduct
to form a protonated Schiff base (species 3), which may be
isolated as a stable covalent species in the presence of
NaCNBH3 (species 4). C, end-labeled
non-adducted DNA or
-HOPdG-adducted DNA (75 nM) was
incubated for 1 h at 4 °C in the absence (lanes 1 and 3-6) or presence (lanes 2 and
7-10) of 50 mM NaCNBH3. Reactions
contained 100 mM NaCl and 1.0 µM (lanes
3 and 7), 10 µM (lanes 4 and
8), 100 µM (lanes 5 and
9), or 1.0 mM (lanes 6 and
10) Lys-Trp-Lys-Lys (KWKK). Reactions were
carried out in the presence of 50 mM HEPES (pH 7.0) and
were quenched by the addition of 100 mM NaBH4
(lanes 2 and 7-10) or H2O
(lanes 1 and 3-6). DNAs were separated through a
denaturing gel as described under "Experimental Procedures," and
the positions of the substrate 12-mer DNAs and the major reduced Schiff
base complexes (12-mer + peptide) are indicated.
-HOPdG adduct by evaluating their propensity to form covalent complexes with Lys-Trp-Lys-Lys in the trapping assay. To directly compare reaction efficiencies, the crotonaldehyde and HNE adducts were
constructed into the same 12-mer sequence as shown for the
-HOPdG
adduct (Fig. 2A), where G* denotes the position of the adducted deoxyguanosine base. Each of the singly adducted oligodeoxynucleotides was individually reacted with Lys-Trp-Lys-Lys in
the presence of NaCNBH3, and the kinetics of complex
formation were evaluated by monitoring the accumulation of a major
DNA-peptide product band on a denaturing gel. At the designated time
points, NaBH4 was added to quench the reaction mixture to
prevent further complex accumulation by facilitating the rapid
reduction of the aldehydic substrate. During a 2-h time course, similar
trapping kinetics were observed for the
-HOPdG adduct and the
crotonaldehyde adducts, and the total amount of trapped complex
observed at the 2-h time point for the (6R,8R)-
and (6S,8S)-crotonaldehyde adducts was virtually
identical at 73 and 71%, respectively (Fig.
3). In contrast, oligodeoxynucleotides
containing the four stereoisomers of the HNE adduct showed significant
differences in reactivity (Fig. 4).
Appreciable complex formation was observed for the
(6S,8R,11S)-HNE adduct by the end of
the 2-h time course (59%) and an intermediate reactivity for the
(6R,8S,11R)-HNE adduct (17%) under
identical reaction conditions. Each of the other two HNE adducts
reacted with poor relative efficiency and showed only 8% complex
formation for the (6R,8S,11S)-HNE
adduct and 7% complex formation for the (6S,8R,11R)-HNE adduct at the 2-h time
point.
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Fig. 3.
Covalent trapping of Lys-Trp-Lys-Lys at the
1,N2-deoxyguanosine adducts of acrolein
and crotonaldehyde. A, reaction mixtures contained 1.0 mM Lys-Trp-Lys-Lys (KWKK) and end-labeled DNA
(75 nM) containing the -HOPdG adduct or the
(6R,8R)- or
(6S,8S)-crotonaldehyde adduct as shown. Reactions
were incubated at 4 °C in the presence of 50 mM HEPES
(pH 7.0), 100 mM NaCl, and 50 mM
NaCNBH3 for 0, 15, 30, 60, 90, and 120 min, followed by
quenching by the addition of 100 mM NaBH4. The
positions of the substrate 12-mer DNAs and the major reduced Schiff
base complexes (12-mer + peptide) are indicated. B, the
kinetics of trapped complex formation are plotted over a 2-h time
course for the
-HOPdG adduct (
) and the
(6R,8R) (
)- and (6S,8S)
(
)-crotonaldehyde adducts.
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Fig. 4.
Covalent trapping of Lys-Trp-Lys-Lys at the
1,N2-deoxyguanosine adducts of HNE.
A, reaction mixtures contained 1.0 mM
Lys-Trp-Lys-Lys (KWKK) and end-labeled DNA (75 nM) containing the
(6R,8S,11R)-,
(6S,8R,11S)-,
(6R,8S,11S)-, or
(6S,8R,11R)-HNE adduct as shown.
Reactions were incubated at 4 °C in the presence of 50 mM HEPES (pH 7.0), 100 mM NaCl, and 50 mM NaCNBH3 for 0, 15, 30, 60, 90, and 120 min
and were quenched by the addition of 100 mM
NaBH4. The positions of the substrate 12-mer DNAs and the
major reduced Schiff base complexes (12-mer + peptide) are indicated.
B, the kinetics of trapped complex formation are plotted
over a 2-h time course for the
(6R,8S,11R) ( )-,
(6S,8R,11S) (
)-,
(6R,8S,11S) (
)-, and
(6S,8R,11R) (
)-HNE adducts.
-HOPdG adduct, the (6R,8R)- and
(6S,8S)-crotonaldehyde adducts, and the
(6S,8R,11S)-HNE adduct. In each case,
preincubation of substrate DNA with NaBH4 dramatically
inhibited the subsequent formation of covalent DNA-peptide cross-links
in a standard trapping assay with Lys-Trp-Lys-Lys and
NaCNBH3 (Fig. 5). In
addition, virtually no shifted complex was observed in the absence of
any reducing agent for each of the aldehyde-adducted DNAs, consistent with the result obtained for the
-HOPdG adduct. From these combined results, we concluded that the formation of a Schiff base mediates covalent attachment of the Lys-Trp-Lys-Lys peptide to the
acrolein-derived
-HOPdG adduct and the crotonaldehyde- and
HNE-derived deoxyguanosine adducts.
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Fig. 5.
Inhibition of complex formation by
pre-reduction of adducted DNAs. Reaction mixtures contained
end-labeled DNA (75 nM) containing the -HOPdG adduct,
the (6S,8R,11S)-HNE adduct, or the
(6R,8R)- or
(6S,8S)-crotonaldehyde adduct as shown. Adducted
DNAs were preincubated in the presence of 75 mM
NaBH4 for 1 h at 4 °C (lanes 2,
3, and 6). Following pretreatment with
NaBH4, DNAs were incubated with 1.0 mM
Lys-Trp-Lys-Lys (KWKK; lanes 4-6) in the absence
(lanes 1, 2, and 4) or presence
(lanes 3, 5, and 6) of 50 mM NaCNBH3. All reactions were carried out in
the presence of 50 mM HEPES (pH 7.0) and 100 mM
NaCl at 4 °C for 1 h. Reactions were quenched by the addition
of 100 mM NaBH4 (lanes 3,
5, and 6) or H2O (lanes 1,
2, and 4). The positions of the substrate 12-mer
DNAs and the major reduced Schiff base complexes (12-mer + peptide) are
indicated.
-HOPdG adduct
showed that this adduct assumes a ring-open structure in duplex DNA (30); however, it was also reported in that study that the
-HOPdG deoxynucleoside exists primarily in the ring-closed conformation. This
difference in tautomer formation prompted an investigation into the
relative reactivity of single- versus double-stranded adducted oligodeoxynucleotides because the rate of formation of DNA-peptide cross-links should be dependent on the concentration of the
ring-open aldehyde adduct under our reaction conditions. In particular,
if the ring-closed tautomer predominates in single-stranded DNA for the
-HOPdG adduct (Fig. 2B, species 2), and the
ring-open aldehyde predominates in duplex DNA (species 1), a
slower rate of DNA-peptide complex formation should be observed in our
trapping assay for the single-stranded
-HOPdG-adducted substrate
versus the duplex substrate. Using the same 12-mer sequence
context as shown in Fig. 2A, we observed that the
double-stranded
-HOPdG-adducted DNA formed 88% complex by the end
of a 2-h time course, whereas the single-stranded DNA formed only 29%
complex (Fig. 6). We next conducted a
similar comparison for the (6R,8R)- and
(6S,8S)-crotonaldehyde adducts and the
(6S,8R,11S)-HNE adduct; and in each
case, the single-stranded adducted DNAs reacted with much slower
reaction kinetics compared with duplex DNAs (Fig. 6). From these
combined results, we concluded that the ring-open aldehyde tautomer is stabilized upon formation of duplex DNA and that the concentration of
ring-open aldehyde is greater in duplex DNA than in single-stranded DNA
for each of the adducts tested.
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Fig. 6.
Lys-Trp-Lys-Lys trapping on single-
versus double-stranded adducted DNAs. Reaction
mixtures contained end-labeled DNA (75 nM) containing the
-HOPdG adduct, the (6S,8R,11S)-HNE
adduct, or the (6R,8R)- or
(6S,8S)-crotonaldehyde adduct as shown.
Single-stranded and duplex substrates were prepared as described under
"Experimental Procedures." Reactions were incubated at 4 °C in
the presence of 50 mM HEPES (pH 7.0), 100 mM
NaCl, and 50 mM NaCNBH3 for 0, 15, 30, 60, 90, and 120 min and were quenched by the addition of 100 mM
NaBH4. The kinetics of trapped complex formation are
plotted over a 2-h time course for the
-HOPdG adduct
(single-stranded (
) and duplex (
)), the
(6S,8R,11S)-HNE adduct
(single-stranded (
) and duplex (
)), the
(6R,8R)-crotonaldehyde adduct (single-stranded
(
) and duplex (
)), and the
(6S,8S)-crotonaldehyde adduct (single-stranded
(
) and duplex (
)).
-elimination at
abasic sites in DNA, such as Lys-Trp-Lys, initiate this chemistry via
the formation of a Schiff base between a peptide amine and C-1' on the
deoxyribose sugar (38, 39). In a recent study, we demonstrated that
lysine-containing peptides utilize the N-terminal
-amine as the
nucleophile in this reaction (35). Because the formation of a
Schiff base requires a neutral (deprotonated) amine, we concluded that
this result is consistent with the lower intrinsic
pKa of an
-amine versus an
-amine.
Specifically, the concentration of neutral
-amine should be far
greater than the relative concentration of
-amine at a given
reaction pH for a lysine-containing peptide. This line of reasoning
also predicts that an
-amine should mediate Schiff base formation in
the case of the adducts under study in this work; thus, the location of
the reactive nucleophile for Lys-Trp-Lys-Lys was interrogated by
examining trapping at the (6S,8R,11S)-HNE adduct as a
representative case. To first test whether a lysine residue is
absolutely required for Schiff base formation, Arg-Trp-Arg-Arg was
substituted for Lys-Trp-Lys-Lys in the standard trapping assay with the
(6S,8R,11S)-HNE-adducted duplex DNA.
This peptide was chosen because arginine residues should retain
positively charged side chain moieties under reaction conditions at pH
7.0, mimicking the electrostatic contribution of lysine in DNA binding.
In this experiment, a similar kinetics profile for the accumulation of
the DNA-peptide complex was observed for each peptide, and the total
amounts of trapped complex observed at the 2-h time point for
Lys-Trp-Lys-Lys and Arg-Trp-Arg-Arg were 68 and 81%, respectively
(Fig. 7). To directly evaluate the role
of the N-terminal
-amine in cross-link formation for each peptide,
the Lys-Trp-Lys-Lys and Arg-Trp-Arg-Arg peptides were modified by the
addition of an N
-acetyl moiety. If the
reactive nucleophile was located at the
-amine,
N
-acetylation should prevent Schiff base
formation by generating an amide at the N terminus. For both peptides,
N
-acetylation dramatically inhibited the
formation of a DNA-peptide complex at the
(6S,8R,11S)-HNE-adducted DNA,
confirming the participation of the amino terminus as the reactive
nucleophile (Fig. 7).
View larger version (13K):
[in a new window]
Fig. 7.
Probing the reactive nucleophile in peptide
trapping for the
(6S,8R,11S)-HNE
adduct. Reaction mixtures contained end-labeled DNA (75 nM) containing the
(6S,8R,11S)-HNE adduct and 1.0 mM Lys-Trp-Lys Lys (KWKK) or 1.0 mM
Arg-Trp-Arg-Arg (RWRR) as shown. Peptides retained either a
free N terminus or an acetylated (N -acetyl) N
terminus. Reactions were incubated at 4 °C in the presence of 50 mM HEPES (pH 7.0), 100 mM NaCl, and 50 mM NaCNBH3 for 0, 15, 30, 60, 90, and 120 min
and were quenched by the addition of 100 mM
NaBH4. The kinetics of trapped complex formation are
plotted over a 2-h time course for Lys-Trp-Lys-Lys (free N terminus
(
) and N
-acetyl terminus (
)) and
Arg-Trp-Arg-Arg (free N terminus (
) and
N
-acetyl terminus (
)).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-erythro-pentofuranosyl)-pyrimido[1,2-a]purin-10(3H)-one (M1G) DNA adduct is a prominent endogenous lesion (44-46),
indicating that malondialdehyde reacts to form a significant number of
DNA adducts in vivo. It is likely that the complexity of
reactions for aldehydes with biomolecules within the cell is not
accurately mimicked by an in vitro examination, particularly
with respect to a multistep reaction leading to the formation of a DPC.
Rather, the spectrum of products observed in vitro provides
insight into the possible reaction pathways for DPC formation, which
may be kinetically favored or disfavored within the cell,
depending on a variety of factors. These combined observations
indicate a multiplicity of pathways that lead to the formation of DPCs
in vivo, one of which may include the formation of stable
aldehyde-derived DNA adducts as reaction intermediates.
-elimination at abasic sites (35). In particular, detection of DNA-peptide complexes by denaturing PAGE necessitated the
stabilization of the cross-links by reduction, and the continuous presence of NaCNBH3 in our reactions irreversibly shifted
the equilibrium toward the accumulation of a reduced Schiff base (Fig. 2B, species 4). As a result, we could not
evaluate the stability of a nonreduced Schiff base complex from the
experiments presented here. However, because we observed a small
detectable level of DNA-peptide complex when reacting Lys-Trp-Lys-Lys
with
-HOPdG in the absence of reducing agent (Fig. 2), we speculate
that the lifetime of a nonreduced Schiff base-mediated DPC is likely
sufficient to interfere with replication or transcription in
vivo.
-amine on the
peptide (Fig. 7), consistent with the lower intrinsic pKa of an
-amine (pKa = 7.6)
compared with an
-amine (pKa = 10.3) in a
random-coil peptide (47). Alternatively, high local concentrations of
positively charged surface residues on a protein may facilitate
reaction of an
-amine on a neighboring lysine because such an
environment will serve to depress the pKa of the
amine. In a previous study, lysine residues were implicated in the
formation of acetaldehyde-induced DPCs (48); and based on the above
reasoning, we suggest that lysines may also react to form Schiff
base-mediated cross-links at the
1,N2-deoxyguanosine adducts under investigation
here. Importantly, such a linkage may represent one of several
possibilities for DPC formation at these adducts because acrolein,
crotonaldehyde, and HNE may also react at histidine and cysteine
residues on proteins (19, 23, 49).
-HOPdG adduct to the ring-open tautomer (30), and a similar result has also been observed for the structurally related
malondialdehyde-derived M1G adduct (50). Consistent with
these earlier observations, the
-HOPdG adduct exhibited faster
reaction kinetics to form DNA-peptide cross-links in duplex DNA
versus single-stranded DNA in our analyses (Fig. 6).
Structural studies have not yet been conducted for the crotonaldehyde
and HNE adducts, although each of the adducts tested gave a result
similar to that obtained with the
-HOPdG adduct in such an
experiment. Thus, we concluded that the formation of duplex DNA most
probably catalyzes ring opening for the (6R,8R)-
and (6S,8S)-crotonaldehyde adducts and the
(6S,8R,11S)-HNE adduct in a fashion
analogous to the
-HOPdG adduct. For duplex oligonucleotides
containing the (6R,8S,11R)-,
(6R,8S,11S)-, and (6S,8R,11R)-HNE adducts, the
observation that these substrates do not readily form cross-links may
indicate that these adducts behave in a chemically distinct fashion
from the (6S,8R,11S)-HNE adduct. It is
as yet unclear how differences in stereochemistry might affect the
ring-open/ring-closed conformational equilibria for these non-reactive
diastereomers. One possibility might be that such adducts exist as
ring-closed tautomers in the syn-conformation about the
glycosidic bond, as shown previously for the
1,N2-propanodeoxyguanosine adduct (51).
Interestingly, the potential for the
-HOPdG adduct to assume a
ring-open structure (capable of forming a canonical Watson-Crick base
pair) has recently led to the proposal that the hydroxylated
-HOPdG
adduct should exhibit lower mutagenicity compared with the
unhydroxylated 1,N2-propanodeoxyguanosine
adduct. In support of this model, in vitro experiments
demonstrated that human polymerase
preferentially incorporates a
cytosine base opposite a
-HOPdG lesion in which the adduct is
trapped as a ring-open structure by reduction, whereas the nonreduced
adduct gives rise to incorporation of adenine and guanine in addition
to cytosine (52). Because the experiments presented here are able to
discriminate between an apparent ring-open versus
ring-closed structure (dependent upon the use of duplex or
single-stranded DNA as a starting substrate), we suggest that peptide
trapping methodology may provide a means to probe the ring structure at
a primer-template junction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Ana M. Sanchez and M. L. Dodson for insightful comments and discussions about this work. We are also grateful to Drs. Irina G. Minko and Paul G. House for helpful discussions and critically reading the manuscript. We thank Stefan Serabyn (NIEHS Center Protein Chemistry Laboratory, University of Texas Medical Branch) for carrying out the peptide synthesis and purification of all peptides used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported in part by NIEHS Award T32 ES07254-10 (to A. J. K.) and Grants P01 ES05355 and P30 ES06676 (to R. S. L.) from the National Institutes of Health.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.
Holds the Distinguished Chair in Environmental Toxicology from the
Houston Endowment. To whom correspondence should be addressed: Sealy
Center for Molecular Science, University of Texas Medical Branch, 5.142 Medical Research Bldg., Galveston, TX 77555-1071. Tel.: 409-772-2179;
Fax: 409-772-1790; E-mail: rslloyd@utmb.edu.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M212012200
2 A. M. Sanchez, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
DPCs, DNA-protein
cross-links;
NER, nucleotide excision repair;
HNE, trans-4-hydroxynonenal;
-HOPdG,
-hydroxy-1,N2-propanodeoxyguanosine
3-(2'-deoxy-
-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine;
HPLC, high performance liquid
chromatography.
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