Acrolein reacts with dG to form hydroxylated 1,N-2-propanodeoxyguanosine (OH-PdG) adducts. Most abundant are the epimeric 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypy rimido[1,2a] purin-10(3H)-ones, commonly referred to as the gamma-OH-PdG adducts. When placed complementary to deoxycytosine in duplex DNA, these undergo rearrangement to the N-2-(3-oxopropyl)-dG aldehyde. The latter forms diastereomeric interstrand N-2-dG:N-2-dG cross-links in the 5'-CpG-3' sequence. Here we report the structure of the stereochemically favored (R)-gamma-hydroxytrimethylene N-2-dG:N-2-dG interstrand DNA cross-link in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3'center dot 5'-d(G(13)G(14)A(15)C(16)T(17)C(18)y(19)C(20)T(21)A(22)G(23)C(24))-3' (X-7 is the dG linked to the alpha-carbon of the carbinolamine linkage, and Y-19 is the dG linked to the gamma-carbon of the carbinolamine linkage; the cross-link is in the 5'-CpG-3' sequence). The structure was characterized using isotope-edited N-15 nuclear Overhauser enhancement spectroscopy heteronuclear single quantum correlation (NOESY-HSQC) NMR, in which the exocyclic amines at X-7 or Y-19 were N-15-labeled. Analyses of NOE intensities involving Y-19 (NH)-H-2 indicated that the (R)-gamma-hydroxytrimethylene linkage was the major cross-link species, constituting 80-90% of the cross-link. The X-7 and Y-11 imino resonances were observed at 65 degrees C. Additionally, for the 5'-neighbor base pair G(5)center dot C-20, the G(5) imino resonance remained sharp at 55 degrees C but broadened at 65 degrees C. In contrast, for the 3'-neighbor A(8)-T-17 base pair, the T-17 imino resonance was severely broadened at 55 degrees C. Structural refinement using NOE distance restraints obtained from isotope-edited N-15 NOESY-HSOC data indicated that the (R)-gamma-hydroxytrimethylene linkage maintained the C-6 center dot Y-19 and X-7 center dot C-18 base pairs with minimal structural perturbations. The (R)-gamma-hydroxytrimethylene linkage was located in the minor groove. The X-7 N-2 and Y-19 N-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-alpha of the tether minimized steric interaction and, more importantly, allowed the formation of a hydrogen bond between the hydroxyl group and C-20 O-2 located in the 5'-neighboring base pair G(5)center dot C-20. 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.