Stereospecific Formation of the (<i>R</i>)-γ-Hydroxytrimethylene Interstrand <i>N</i>²-dG:<i>N</i>²-dG Cross-Link Arising from the γ-OH-1,<i>N</i>²-Propano-2′-deoxyguanosine Adduct in the 5′-CpG-3′ DNA Sequence

Abstract

Acrolein reacts with dG to form hydroxylated 1,<i>N</i>²-propanodeoxyguanosine (OH-PdG) adducts. Most abundant are the epimeric 3-(2-deoxy-β-d-<i>erythro</i>-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2<i>a</i>] purin-10(3<i>H</i>)-ones, commonly referred to as the γ-OH-PdG adducts. When placed complementary to deoxycytosine in duplex DNA, these undergo rearrangement to the <i>N</i>²-(3-oxopropyl)-dG aldehyde. The latter forms diastereomeric interstrand <i>N</i>²-dG:<i>N</i>²-dG cross-links in the 5′-CpG-3′ sequence. Here we report the structure of the stereochemically favored (<i>R</i>)-γ-hydroxytrimethylene <i>N</i>²-dG:<i>N</i>²-dG interstrand DNA cross-link in 5′-d(G¹C²T³A⁴G⁵C⁶X⁷A⁸G⁹T¹⁰C¹¹C¹²)-3′·5′-d(G¹³G¹⁴A¹⁵C¹⁶T¹⁷C¹⁸Y¹⁹C²⁰T²¹A²²G²³C²⁴)-3′ (X⁷ is the dG linked to the α-carbon of the carbinolamine linkage, and Y¹⁹ is the dG linked to the γ-carbon of the carbinolamine linkage; the cross-link is in the 5′-CpG-3′ sequence). The structure was characterized using isotope-edited ¹⁵N nuclear Overhauser enhancement spectroscopy heteronuclear single quantum correlation (NOESY-HSQC) NMR, in which the exocyclic amines at X⁷ or Y¹⁹ were ¹⁵N-labeled. Analyses of NOE intensities involving Y¹⁹ <i>N</i>²H indicated that the (<i>R</i>)-γ-hydroxytrimethylene linkage was the major cross-link species, constituting 80−90% of the cross-link. The X⁷ and Y¹⁹ imino resonances were observed at 65 °C. Additionally, for the 5′-neighbor base pair G⁵·C²⁰, the G⁵ imino resonance remained sharp at 55 °C but broadened at 65 °C. In contrast, for the 3′-neighbor A⁸·T¹⁷ base pair, the T¹⁷ imino resonance was severely broadened at 55 °C. Structural refinement using NOE distance restraints obtained from isotope-edited ¹⁵N NOESY-HSQC data indicated that the (<i>R</i>)-γ-hydroxytrimethylene linkage maintained the C⁶·Y¹⁹ and X⁷·C¹⁸ base pairs with minimal structural perturbations. The (<i>R</i>)-γ-hydroxytrimethylene linkage was 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, which maintained Watson−Crick hydrogen bonding of the cross-linked base pairs. The anti conformation of the hydroxyl group with respect to C^α of the tether minimized steric interaction and, more importantly, allowed the formation of a hydrogen bond between the hydroxyl group and C²⁰ <i>O</i>² located in the 5′-neighboring base pair G⁵·C²⁰. The formation of this hydrogen bond may, in part, explain the thermal stability of this carbinolamine interstrand cross-link and the stereochemical preference for the (<i>R</i>) configuration of the cross-link