Stereospecific Formation of the (R)-γ-HydroxytrimethyIene Interstrand N[sup2]-dG:N[sup2]-dG Cross-Link Arising from the γ-OH-1 ,N[sup2]-Propano-2'-deoxyguanosine Adduct in the 5'-CpG-3' DNA Sequence.

Acrolein reacts with dG to form hydroxylated 1 ,N[sup2]-propanodeoxyguanosine (OH-PdG) adducts. Most abundant are the epimeric 3-(2-deoxy-β-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hyfroxypyrimido[1,2a] purin-10(3H)-ones, commonly referred to as the γ-OH-PdG adducts. When placed complementary to deoxycytosine in duplex DNA, these undergo rearrangement to the N[sup2]-(3-oxopropyl)-dG aldehyde. The latter forms diastereomeric interstrand N[sup2]-dG:N[sup2]-dG cross-links in the 5'-CpG-3' sequence. Here we report the structure of the stereochemically favored (R)-γ-hydroxytrimethylene N[sup2]-dG:M-dG interstrand DNA cross- link in 5'-d(G[sup1] C[sup2]T[sup3]A[sup4]G[sup5]C[sup6]X[sup7]A[sup8]G[sup9]T[sup10]C[sup11]C[sup12])-3'∙ 5'-d(G[sup13]G[sup14]A[sup15]C[sup16]T[sup17]C[sup18]Y[supl9]C[sup20]T[sup21]A[sup22]G[sup23]C[sup24])-3' (X7 is the dG linked to the α-carbon of the carbinolamine linkage, and Y[sup19] 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 [sup15]N nuclear Overhauser enhancement spectroscopy heteronuclear single quantum correlation (NOESY-HSQC) NMR, in which the exocyclic amines at X[sup7] or Y[sup19] were [sup15]N-labeled. Analyses of NOE intensities involving Y[sup19] N[sup2]H indicated that the (R)-γ-hydroxytrimethylene linkage was the major cross-link species, constituting 80-90% of the cross-link. The X[sup7] and Y[sup19] imino resonances were observed at 65 °C. Additionally, for the 5'-neighbor base pair G[sup5]C[sup20], the G[sup5] imino resonance remained sharp at 55 °C but broadened at 65 °C. In contrast, for the 3'-neighbor A[sup8]∙T[sup17] base pair, the T[sup17] 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 (R)-γ-hydroxytrimethylene linkage maintained the C[sup6]∙Y[sup19] and X[sup7]∙C[sup18] base pairs with minimal structural perturbations. The (R)-γ-hydroxytrimethylene linkage was located in the minor groove. The X[sup7] N[sup2] and Y[sup19] N[sup2] 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' of the tether minimized steric interaction and, more importantly, allowed the formation of a hydrogen bond between the hydroxyl group and C[sup20] O[sup2] located in the 5'-neighboring base pair G[sup5]∙C[sup20]. 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 (R) configuration of the cross-link. [ABSTRACT FROM AUTHOR]