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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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Time course of γ-HOPdG cross-linking to EcoRI. γ-HOPdG-modified 32P-end-labeled DNA substrate (160 pM) was incubated with EcoRI (1.6 nM) for up to 5 h at room temperature in the presence of 50 mM NaCNBH3, and then, a second reducing agent, NaBH4 (100 mM), was added to rapidly quench any remaining aldehydic substrate at specific time points. A representative autoradiogram from three independent experiments is presented, which displays the accumulation of the DNA−protein cross-linked band. Percent cross-linking is shown in the graph just below the gel expansion. The values are the means ± standard deviations from three independent experiments.
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fig5: Time course of γ-HOPdG cross-linking to EcoRI. γ-HOPdG-modified 32P-end-labeled DNA substrate (160 pM) was incubated with EcoRI (1.6 nM) for up to 5 h at room temperature in the presence of 50 mM NaCNBH3, and then, a second reducing agent, NaBH4 (100 mM), was added to rapidly quench any remaining aldehydic substrate at specific time points. A representative autoradiogram from three independent experiments is presented, which displays the accumulation of the DNA−protein cross-linked band. Percent cross-linking is shown in the graph just below the gel expansion. The values are the means ± standard deviations from three independent experiments.

Mentions: Cross-link formation was monitored over 5 h (Figure 5). Reactions were prepared in the presence of NaCNBH3, and then, a second reducing agent, NaBH4, was added to rapidly quench any remaining aldehydic substrate at specific time points. The γ-HOPdG−EcoRI cross-link was observed within 10 min. The conversion of free DNA to cross-linked product reached a plateau within 3 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)

Time course of γ-HOPdG cross-linking to EcoRI. γ-HOPdG-modified 32P-end-labeled DNA substrate (160 pM) was incubated with EcoRI (1.6 nM) for up to 5 h at room temperature in the presence of 50 mM NaCNBH3, and then, a second reducing agent, NaBH4 (100 mM), was added to rapidly quench any remaining aldehydic substrate at specific time points. A representative autoradiogram from three independent experiments is presented, which displays the accumulation of the DNA−protein cross-linked band. Percent cross-linking is shown in the graph just below the gel expansion. The values are the means ± standard deviations from three independent experiments.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651693&req=5

fig5: Time course of γ-HOPdG cross-linking to EcoRI. γ-HOPdG-modified 32P-end-labeled DNA substrate (160 pM) was incubated with EcoRI (1.6 nM) for up to 5 h at room temperature in the presence of 50 mM NaCNBH3, and then, a second reducing agent, NaBH4 (100 mM), was added to rapidly quench any remaining aldehydic substrate at specific time points. A representative autoradiogram from three independent experiments is presented, which displays the accumulation of the DNA−protein cross-linked band. Percent cross-linking is shown in the graph just below the gel expansion. The values are the means ± standard deviations from three independent experiments.
Mentions: Cross-link formation was monitored over 5 h (Figure 5). Reactions were prepared in the presence of NaCNBH3, and then, a second reducing agent, NaBH4, was added to rapidly quench any remaining aldehydic substrate at specific time points. The γ-HOPdG−EcoRI cross-link was observed within 10 min. The conversion of free DNA to cross-linked product reached a plateau within 3 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