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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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Related in: MedlinePlus

γ-HOPdG forms a cross-link to EcoRI. 32P-labeled γ-HOPdG- or M1dG-modified or unmodified DNA substrates (160 pM) were incubated with EcoRI (1.6 nM) at room temperature for 2 h in the presence or absence of 50 mM NaCNBH3. The products were resolved by SDS-PAGE and visualized by phosphorimager analysis. This is a representative autoradiogram from at least five independent experiments. The cross-linked product is indicated by the arrow.
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fig2: γ-HOPdG forms a cross-link to EcoRI. 32P-labeled γ-HOPdG- or M1dG-modified or unmodified DNA substrates (160 pM) were incubated with EcoRI (1.6 nM) at room temperature for 2 h in the presence or absence of 50 mM NaCNBH3. The products were resolved by SDS-PAGE and visualized by phosphorimager analysis. This is a representative autoradiogram from at least five independent experiments. The cross-linked product is indicated by the arrow.

Mentions: EcoRI binds the palindromic sequence, 5′-GAATTC-3′, and sequentially cleaves both strands of the DNA duplex at the phosphodiester bond positioned 3′ to deoxyguanosine. 32P-labeled oligonucleotide duplexes were prepared containing a unique EcoRI recognition site that was modified with γ-HOPdG. Substrate DNA was incubated for 2 h with EcoRI (molar ratio of DNA duplex: EcoRI = 1:10) in the presence of NaCNBH3, as described in the . NaCNBH3 does not react with EcoRI or γ-HOPdG but is capable of reducing an imine cross-link between the free aldehyde form of γ-HOPdG and a lysine residue. EcoRI reacted with γ-HOPdG-modified substrates to form a DNA−protein cross-link observable by SDS-PAGE analysis (Figure 2). Formation of the gel-shifted band corresponding to the protein−DNA complex was dependent on stabilization of the cross-link by reduction with NaCNBH3. A weaker, second band was observed that migrated more slowly than the major γ-HOPdG−EcoRI band. The identity of this second band is unknown.


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

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

γ-HOPdG forms a cross-link to EcoRI. 32P-labeled γ-HOPdG- or M1dG-modified or unmodified DNA substrates (160 pM) were incubated with EcoRI (1.6 nM) at room temperature for 2 h in the presence or absence of 50 mM NaCNBH3. The products were resolved by SDS-PAGE and visualized by phosphorimager analysis. This is a representative autoradiogram from at least five independent experiments. The cross-linked product is indicated by the arrow.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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

fig2: γ-HOPdG forms a cross-link to EcoRI. 32P-labeled γ-HOPdG- or M1dG-modified or unmodified DNA substrates (160 pM) were incubated with EcoRI (1.6 nM) at room temperature for 2 h in the presence or absence of 50 mM NaCNBH3. The products were resolved by SDS-PAGE and visualized by phosphorimager analysis. This is a representative autoradiogram from at least five independent experiments. The cross-linked product is indicated by the arrow.
Mentions: EcoRI binds the palindromic sequence, 5′-GAATTC-3′, and sequentially cleaves both strands of the DNA duplex at the phosphodiester bond positioned 3′ to deoxyguanosine. 32P-labeled oligonucleotide duplexes were prepared containing a unique EcoRI recognition site that was modified with γ-HOPdG. Substrate DNA was incubated for 2 h with EcoRI (molar ratio of DNA duplex: EcoRI = 1:10) in the presence of NaCNBH3, as described in the . NaCNBH3 does not react with EcoRI or γ-HOPdG but is capable of reducing an imine cross-link between the free aldehyde form of γ-HOPdG and a lysine residue. EcoRI reacted with γ-HOPdG-modified substrates to form a DNA−protein cross-link observable by SDS-PAGE analysis (Figure 2). Formation of the gel-shifted band corresponding to the protein−DNA complex was dependent on stabilization of the cross-link by reduction with NaCNBH3. A weaker, second band was observed that migrated more slowly than the major γ-HOPdG−EcoRI band. The identity of this second band is unknown.

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