fig7: EcoRI cleavage following reduction of the cross-link. 32P-radiolabeled γ-HOPdG-modified substrate or unmodified substrate (500 nM) was incubated with EcoRI (5 nM) in the absence of Mg2+ and either the presence or the absence of NaCNBH3 for 2 h. Following preincubation, MgCl2 (10 mM) was added to initiate cleavage. Reactions were incubated at 37 °C for various times. Cleavage profiles shown are for the 21-mer strand of the DNA duplex. Unmodified substrate, −NaCNBH3 (◼); unmodified substrate, +NaCNBH3 (◻); γ-HOPdG-modified substrate, −NaCNBH3 (●); and γ-HOPdG-modified substrate, +NaCNBH3 incubation (○).

Mentions: To examine whether a stabilized cross-link could affect EcoRI activity, γ-HOPdG-modified or unmodified substrates were incubated with EcoRI in the absence of Mg2+ and either the presence or the absence of NaCNBH3 for 2 h; then, MgCl2 was added to the reaction mixture to initiate cleavage. Following MgCl2 addition, EcoRI cleavage of the substrate was substantially decreased in samples containing γ-HOPdG-modified DNA and NaCNBH3 as compared to samples containing γ-HOPdG-modified substrate in the absence of reducing agent. There was no effect of NaCNBH3 on cleavage of the unmodified DNA substrate by EcoRI (Figure 7).

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.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.

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.