Mechanism of Repair of Acrolein- and Malondialdehyde-Derived Exocyclic Guanine Adducts by the α-Ketoglutarate/Fe(II) Dioxygenase AlkB

The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M[subscript 1]dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M[subscript 1]dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M[subscript 1]dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH[subscript 4]. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.National Institutes of Health (U.S.) (Grant R01 CA080024)National Institutes of Health (U.S.) (Grant R01 CA26731)National Institutes of Health (U.S.) (Center Grant P30 ES02109)National Institutes of Health (U.S.) (Training Grant T32 ES007020

The modulation of topoisomerase I-mediated DNA

cleavage and the induction of DNA–topoisomerase I crosslinks by crotonaldehyde-derived DNA adduct

Formation of a <i>N</i>²-dG:<i>N</i>²-dG Carbinolamine DNA Cross-link by the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG Adduct

Michael addition of <i>trans</i>-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,<i>N</i>²-dG adducts in DNA. When placed opposite dC in the 5′-CpG-3′ sequence, the (6<i>S</i>,8<i>R</i>,11<i>S</i>) diastereomer forms a <i>N</i>²-dG:<i>N</i>²-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. <i>J. Am. Chem. Soc.</i> <b>2003</b>, <i>125</i>, 5687–5700]. We refined its structure in 5′-d(G¹C²T³A⁴G⁵C⁶<u>X</u>⁷A⁸G⁹T¹⁰C¹¹C¹²)-3′·5′-d(G¹³G¹⁴A¹⁵C¹⁶T¹⁷C¹⁸<u>Y</u>¹⁹C²⁰T²¹A²²G²³C²⁴)-3′ [X⁷ is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct, and Y¹⁹ is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct; the cross-link is in the 5′-CpG-3′ sequence]. Introduction of ¹³C at the C8 carbon of the cross-link revealed one ¹³C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y¹⁹ H1′, C²⁰ H1′, and C²⁰ H4′, orienting it toward the complementary strand, consistent with the (6<i>S</i>,8<i>R</i>,11<i>S</i>) configuration. An NOE was also observed between the HNE H11 proton and Y¹⁹ H1′, orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y¹⁹ <i>N</i>²H and X⁷ N1H protons, respectively. A strong H8→H11 NOE and no ³<i>J</i>(¹³C→H) coupling for the ¹³C8–O–C11–H11 eliminated the tetrahydrofuran species derived from the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct. The (6<i>S</i>,8<i>R</i>,11<i>S</i>) carbinolamine linkage and the HNE side chain were located in the minor groove. The X⁷ <i>N</i>² and Y¹⁹ <i>N</i>² atoms were in the gauche conformation with respect to the linkage, maintaining Watson–Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C²⁰ <i>O</i>² of the 5′-neighbor base pair G⁵·C²⁰ and O11H with C¹⁸ <i>O</i>² of X⁷·C¹⁸. These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6<i>S</i>,8<i>R</i>,11<i>S</i>) configuration