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Formation of DNA-protein cross-links between gamma-hydroxypropanodeoxyguanosine and EcoRI.

VanderVeen LA, Harris TM, Jen-Jacobson L, Marnett LJ - Chem. Res. Toxicol. (2008)

Bottom Line: Interestingly, the cross-link did not restrict the ability of EcoRI to cleave DNA substrates.However, stabilization of the cross-link by reduction of the Schiff base linkage resulted in loss of enzyme activity.Reversal of cross-link formation allows EcoRI to effect enzymatic cleavage of competitor oligonucleotides.

View Article: PubMed Central - PubMed

Affiliation: A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

ABSTRACT
The toxicity of acrolein, an alpha,beta-unsaturated aldehyde produced during lipid peroxidation, is attributable to its high reactivity toward DNA and cellular proteins. The major acrolein-DNA adduct, gamma-hydroxypropano-2'-deoxyguanosine (gamma-HOPdG), ring opens to form a reactive N(2)-oxopropyl moiety that cross-links to DNA and proteins. We demonstrate the ability of gamma-HOPdG in a duplex oligonucleotide to cross-link to a protein (EcoRI) that specifically interacts with DNA at a unique sequence. The formation of a cross-link to EcoRI was dependent on the intimate binding of the enzyme to its gamma-HOPdG-modified recognition site. Interestingly, the cross-link did not restrict the ability of EcoRI to cleave DNA substrates. However, stabilization of the cross-link by reduction of the Schiff base linkage resulted in loss of enzyme activity. This work indicates that the gamma-HOPdG-EcoRI cross-link is in equilibrium with free oligonucleotide and enzyme. Reversal of cross-link formation allows EcoRI to effect enzymatic cleavage of competitor oligonucleotides.

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Effect of γ-HOPdG on EcoRI activity. (A) 5′-Radiolabeled duplex containing a single EcoRI recognition site. The arrows depict cleavage sites. γ-HOPdG was contained on the 21-mer strand, as indicated by “N”. (B) Cleavage reactions containing 5 nM 32P-labeled oligonucleotide duplex, 495 nM nonlabeled oligonucleotide substrate, and 5 nM EcoRI were incubated at 37 °C for various times, then quenched, and subjected to gel electrophoresis and phosphorimager analysis as described in the . Unmodified substrate, 27-mer strand (◼); γ-HOPdG-modified substrate, 27-mer strand (▼); γ-HOPdG-modified substrate, 21-mer strand (▲). (C) Nonlabeled oligonucleotide duplexes modified with γ-HOPdG or unmodified duplexes (500 nM) were incubated with EcoRI endonuclease (5 nM) for 2 h at 37 °C, prior to the addition of fresh unmodified EcoRI substrate, spiked with 32P-labeled unmodified substrate, to a final concentration of 500 nM. Cleavage of the radiolabeled substrate was monitored for 2 h. The data shown represent cleavage of the 27-mer strand of the radiolabeled duplex in the presence of the indicated competitor substrate. The 21-mer strand of each duplex was cleaved similarly to its complement (data not shown). Unmodified competitor substrate (◼); γ-HOPdG-modified competitor substrate (▲). The values are the means ± standard deviations from three independent experiments.
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fig6: Effect of γ-HOPdG on EcoRI activity. (A) 5′-Radiolabeled duplex containing a single EcoRI recognition site. The arrows depict cleavage sites. γ-HOPdG was contained on the 21-mer strand, as indicated by “N”. (B) Cleavage reactions containing 5 nM 32P-labeled oligonucleotide duplex, 495 nM nonlabeled oligonucleotide substrate, and 5 nM EcoRI were incubated at 37 °C for various times, then quenched, and subjected to gel electrophoresis and phosphorimager analysis as described in the . Unmodified substrate, 27-mer strand (◼); γ-HOPdG-modified substrate, 27-mer strand (▼); γ-HOPdG-modified substrate, 21-mer strand (▲). (C) Nonlabeled oligonucleotide duplexes modified with γ-HOPdG or unmodified duplexes (500 nM) were incubated with EcoRI endonuclease (5 nM) for 2 h at 37 °C, prior to the addition of fresh unmodified EcoRI substrate, spiked with 32P-labeled unmodified substrate, to a final concentration of 500 nM. Cleavage of the radiolabeled substrate was monitored for 2 h. The data shown represent cleavage of the 27-mer strand of the radiolabeled duplex in the presence of the indicated competitor substrate. The 21-mer strand of each duplex was cleaved similarly to its complement (data not shown). Unmodified competitor substrate (◼); γ-HOPdG-modified competitor substrate (▲). The values are the means ± standard deviations from three independent experiments.

Mentions: Oligonucleotide duplexes containing a single adduct located within a unique EcoRI recognition site were 5′-32P-end-labeled on both strands. The adducted strand of the duplex was a different length than its complement, and the oligonucleotides were designed to release 32P-end-labeled cleavage products of different lengths. Thus, simultaneous monitoring of cleavage of both strands of the duplex was achieved. The complement strand of the γ-HOPdG-modified substrate was cleaved nearly as efficiently as unmodified substrate (∼90%) (Figure 6B). In contrast, only ∼20% of the γ-HOPdG-modified strand was cleaved by EcoRI within 2 h.


Formation of DNA-protein cross-links between gamma-hydroxypropanodeoxyguanosine and EcoRI.

VanderVeen LA, Harris TM, Jen-Jacobson L, Marnett LJ - Chem. Res. Toxicol. (2008)

Effect of γ-HOPdG on EcoRI activity. (A) 5′-Radiolabeled duplex containing a single EcoRI recognition site. The arrows depict cleavage sites. γ-HOPdG was contained on the 21-mer strand, as indicated by “N”. (B) Cleavage reactions containing 5 nM 32P-labeled oligonucleotide duplex, 495 nM nonlabeled oligonucleotide substrate, and 5 nM EcoRI were incubated at 37 °C for various times, then quenched, and subjected to gel electrophoresis and phosphorimager analysis as described in the . Unmodified substrate, 27-mer strand (◼); γ-HOPdG-modified substrate, 27-mer strand (▼); γ-HOPdG-modified substrate, 21-mer strand (▲). (C) Nonlabeled oligonucleotide duplexes modified with γ-HOPdG or unmodified duplexes (500 nM) were incubated with EcoRI endonuclease (5 nM) for 2 h at 37 °C, prior to the addition of fresh unmodified EcoRI substrate, spiked with 32P-labeled unmodified substrate, to a final concentration of 500 nM. Cleavage of the radiolabeled substrate was monitored for 2 h. The data shown represent cleavage of the 27-mer strand of the radiolabeled duplex in the presence of the indicated competitor substrate. The 21-mer strand of each duplex was cleaved similarly to its complement (data not shown). Unmodified competitor substrate (◼); γ-HOPdG-modified competitor substrate (▲). The values are the means ± standard deviations from three independent experiments.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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fig6: Effect of γ-HOPdG on EcoRI activity. (A) 5′-Radiolabeled duplex containing a single EcoRI recognition site. The arrows depict cleavage sites. γ-HOPdG was contained on the 21-mer strand, as indicated by “N”. (B) Cleavage reactions containing 5 nM 32P-labeled oligonucleotide duplex, 495 nM nonlabeled oligonucleotide substrate, and 5 nM EcoRI were incubated at 37 °C for various times, then quenched, and subjected to gel electrophoresis and phosphorimager analysis as described in the . Unmodified substrate, 27-mer strand (◼); γ-HOPdG-modified substrate, 27-mer strand (▼); γ-HOPdG-modified substrate, 21-mer strand (▲). (C) Nonlabeled oligonucleotide duplexes modified with γ-HOPdG or unmodified duplexes (500 nM) were incubated with EcoRI endonuclease (5 nM) for 2 h at 37 °C, prior to the addition of fresh unmodified EcoRI substrate, spiked with 32P-labeled unmodified substrate, to a final concentration of 500 nM. Cleavage of the radiolabeled substrate was monitored for 2 h. The data shown represent cleavage of the 27-mer strand of the radiolabeled duplex in the presence of the indicated competitor substrate. The 21-mer strand of each duplex was cleaved similarly to its complement (data not shown). Unmodified competitor substrate (◼); γ-HOPdG-modified competitor substrate (▲). The values are the means ± standard deviations from three independent experiments.
Mentions: Oligonucleotide duplexes containing a single adduct located within a unique EcoRI recognition site were 5′-32P-end-labeled on both strands. The adducted strand of the duplex was a different length than its complement, and the oligonucleotides were designed to release 32P-end-labeled cleavage products of different lengths. Thus, simultaneous monitoring of cleavage of both strands of the duplex was achieved. The complement strand of the γ-HOPdG-modified substrate was cleaved nearly as efficiently as unmodified substrate (∼90%) (Figure 6B). In contrast, only ∼20% of the γ-HOPdG-modified strand was cleaved by EcoRI within 2 h.

Bottom Line: Interestingly, the cross-link did not restrict the ability of EcoRI to cleave DNA substrates.However, stabilization of the cross-link by reduction of the Schiff base linkage resulted in loss of enzyme activity.Reversal of cross-link formation allows EcoRI to effect enzymatic cleavage of competitor oligonucleotides.

View Article: PubMed Central - PubMed

Affiliation: A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

ABSTRACT
The toxicity of acrolein, an alpha,beta-unsaturated aldehyde produced during lipid peroxidation, is attributable to its high reactivity toward DNA and cellular proteins. The major acrolein-DNA adduct, gamma-hydroxypropano-2'-deoxyguanosine (gamma-HOPdG), ring opens to form a reactive N(2)-oxopropyl moiety that cross-links to DNA and proteins. We demonstrate the ability of gamma-HOPdG in a duplex oligonucleotide to cross-link to a protein (EcoRI) that specifically interacts with DNA at a unique sequence. The formation of a cross-link to EcoRI was dependent on the intimate binding of the enzyme to its gamma-HOPdG-modified recognition site. Interestingly, the cross-link did not restrict the ability of EcoRI to cleave DNA substrates. However, stabilization of the cross-link by reduction of the Schiff base linkage resulted in loss of enzyme activity. This work indicates that the gamma-HOPdG-EcoRI cross-link is in equilibrium with free oligonucleotide and enzyme. Reversal of cross-link formation allows EcoRI to effect enzymatic cleavage of competitor oligonucleotides.

Show MeSH
Related in: MedlinePlus