Biochemical effects of 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), an abasic lesion resulting from the oxidation of DNA and characterization of 5-ethynyl-2'-deoxycytidine as a mechanistic probe.

摘要

The oxidized abasic lesion, 5'-(2-phosphory1-1,4-dioxobutane) (DOB), is produced in DNA as a result of cleavage of the C4'-05' carbon-carbon bond following hydrogen atom abstraction from the C5'-position. DOB is produced by a variety of DNA-damaging agents, including γ-radiolysis and antitumor agents, such as the enediynes. Using ternary complexes in which DOB is produced from a synthetic precursor, we show that the lesion forms interstrand cross-links (ICLs) selectively with a dA opposite the 3'-adjacent nucleotide. Product studies and the use of monoaldehyde models suggest that ICL formation involves condensation of the dialdehyde with the exocyclic amine Kinetic studies indicate that in the ternary complex, DOB produces cross-links reversibly and undergoes 3-elimination to give 1,4-butene-2-dial.;Repair of DOB is important for maintaining the integrity of the genome. We demonstrate that DOB efficiently and irreversibly inhibits the lyase activity of polymerase β (Pol β), a DNA base excision repair (BER) enzyme. The phenol extraction of the reactions between radiolabeled DNA containing DOB and Pol β revealed that the inhibition mostly results from the formation of DNA protein cross-links (DPCs). GluC and tryptic digestion of the mixture of Pol β and DOB suggest that the lysine residues in the lyase active site of Pol β are alkylated by DOB or the elimination product of DOB, 1,4-butene-2-dial. The repair of DOB does not follow short-patch or long-patch BER. The extension reaction by Pol β is greatly suppressed by DOB.;The elimination product of DOB is 1,4-butene-2-dial, which is also a metabolite of furan. Using site-specific generation of 3H-DOB, exocyclic DNA adducts arising from DOB were detected. The dG adduct resulting from the DOB elimination product, 1,4-butene-2-dial was detected in DNA for the first time using the method presented here. Our results suggest that the 5' oxidative DNA lesion is also a significant source of 1,4-butene-2-dial and contributes to the likely mutagenesis resulting from this α,β-unsaturated bis-electrophile.;Cytosine methylation of genomic DNA is a fundamental epigenetic mechanism for regulating the integrity of mammalian genomes and gene expression. The active DNA demethylation (DdM), reversal of DNA methylation, can be accomplished via a base excision repair (BER) pathway. A mechanistic probe, 5-ethyny1-2'-deoxycytidine (EdC), can be incorporated into the DNA sequence, which undergoes active DNA demethylation via BER. Kinetic studies suggested that the triphosphate of MC was efficiently incorporated into DNA by the BER enzyme, Pol β. The reaction between fluorescein azide and DNA containing EdC suggested that the conjugation of EdC with fluorophore azide can be used to visualize the DNA undergoing active DNA demethylation.