1,N2-Deoxyguanosine Adducts of Acrolein, Crotonaldehyde, and trans-4-Hydroxynonenal Cross-link to Peptides via Schiff Base Linkage*

Andrew J. Kurtz and R. Stephen LloydDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 gamma -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 Nalpha -acetylation of peptides dramatically inhibited trapping; thus, the reactive nucleophile is located at the N-terminal alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, gamma -hydroxy-1,N2-propanodeoxyguanosine (gamma -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 gamma -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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. [gamma -32P]ATP was obtained from PerkinElmer Life Sciences.

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-1 cm-1 as the Trp molar extinction coefficient (36).

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 gamma -32P-labeled on the 5'-end with T4 polynucleotide kinase following standard procedures.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Covalent Trapping of Lys-Trp-Lys-Lys at the gamma -HOPdG Adduct-- A prior NMR study on the gamma -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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -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.

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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -HOPdG adduct (black-square) and the (6R,8R) (triangle )- and (6S,8S) (black-triangle)-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) (black-square)-, (6S,8R,11S) ()-, (6R,8S,11S) (black-triangle)-, and (6S,8R,11R) (triangle )-HNE adducts.

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 gamma -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 gamma -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 gamma -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 gamma -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.

Reactivity of Single- Versus Double-stranded Aldehyde-adducted DNAs-- As stated above, a prior NMR study of the gamma -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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -HOPdG adduct (single-stranded (triangle ) and duplex (black-triangle)), the (6S,8R,11S)-HNE adduct (single-stranded (down-triangle) and duplex (black-down-triangle )), the (6R,8R)-crotonaldehyde adduct (single-stranded (open circle ) and duplex ()), and the (6S,8S)-crotonaldehyde adduct (single-stranded () and duplex (black-square)).

Investigation of the Reactive Nucleophile-- Peptides capable of catalyzing beta -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 alpha -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 alpha -amine versus an epsilon -amine. Specifically, the concentration of neutral alpha -amine should be far greater than the relative concentration of epsilon -amine at a given reaction pH for a lysine-containing peptide. This line of reasoning also predicts that an alpha -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 alpha -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 Nalpha -acetyl moiety. If the reactive nucleophile was located at the alpha -amine, Nalpha -acetylation should prevent Schiff base formation by generating an amide at the N terminus. For both peptides, Nalpha -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).


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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 (Nalpha -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 (black-triangle) and Nalpha -acetyl terminus (triangle )) and Arg-Trp-Arg-Arg (free N terminus (black-square) and Nalpha -acetyl terminus ()).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.

The borohydride trapping methodology employed in this study was utilized previously to isolate Schiff base intermediates in peptide-catalyzed beta -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 gamma -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.

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 alpha -amine on the peptide (Fig. 7), consistent with the lower intrinsic pKa of an alpha -amine (pKa = 7.6) compared with an epsilon -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 epsilon -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).

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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -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 gamma -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 eta  preferentially incorporates a cytosine base opposite a gamma -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.

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.

    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.

Dagger 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; gamma -HOPdG, gamma -hydroxy-1,N2-propanodeoxyguanosine 3-(2'-deoxy-beta -D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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