Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis. MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine. In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F. H. and Goldstein, B. D. Science 1976, 191, 868--869). The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines. An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions. The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link. The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation. This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.

Malondialdehyde interstrand cross-links in DNA show strong preference for 5'-d(CpG) sequences. The cross-links are unstable and a trimethylene cross-link has been used as a surrogate for structural studies. A previous structural study of the 5'-d(CpG) cross-link in the sequence 5'-d(AGGCGCCT), where G is the modified nucleotide, by NMR spectroscopy and molecular dynamics using a simulated annealing protocol showed the guanine residues and the tether lay approximately in a plane such that the trimethylene tether and probably the malondialdehyde tether, as well, could be accommodated without major disruptions of duplex structure [Dooley et al. J. Am Chem. Soc. 2001, 123, 1730-1739]. The trimethylene cross-link has now been studied in a GpC motif using the reverse sequence. The structure lacks the planarity seen with the 5'-d(CpG) sequence and is skewed about the trimethylene cross-link. Melting studies indicate that the trimethylene cross-link is thermodynamically less stable in the GpC motif than in the 5-d(CpG). Furthermore, lack of planarity of the GpC cross-link precludes making an isosteric replacement of the trimethylene tether by malondialdehyde. A similar argument can be used to explain the 5'-d(CpG) preference for interchain cross-linking by acrolein.

Synthetically derived trimethylene interstrand DNA cross-links have been used as surrogates for the native cross-links that arise from the 1,N2-deoxyguanosine adducts derived from α,β-unsaturated aldehydes. The native enal-mediated cross-linking occurs in the 5′-CpG-3′ sequence context but not in the 5′-GpC-3′ sequence context. The ability of the native enal-derived 1,N2-dG adducts to induce interstrand DNA cross-links in the 5′-CpG-3′ sequence as opposed to the 5′-GpC-3′ sequence is attributed to the destabilization of the DNA duplex in the latter sequence context. Here, we report higher accuracy solution structures of the synthetically derived trimethylene cross-links, which are refined from NMR data with the AMBER force field. When the synthetic trimethylene cross-links are placed into either the 5′-CpG-3′ or the 5′-GpC-3′ sequence contexts, the DNA duplex maintains B-DNA geometry with structural perturbations confined to the cross-linked base pairs. Watson−Crick hydrogen bonding is conserved throughout the duplexes. Although different from canonical B-DNA stacking, the cross-linked and the neighbor base pairs stack in the 5′-CpG-3′ sequence. In contrast, the stacking at the cross-linked base pairs in the 5′-GpC-3′ sequence is greatly perturbed. The π-stacking interactions between the cross-linked and the neighbor base pairs are reduced. This is consistent with remarkable chemical shift perturbations of the C5 H5 and H6 nucleobase protons that shifted downfield by 0.4−0.5 ppm. In contrast, these chemical shift perturbations in the 5′-CpG-3′ sequence are not remarkable, consistent with the stacked structure. The differential stacking of the base pairs at the cross-linking region probably explains the difference in stabilities of the trimethylene cross-links in the 5′-CpG-3′ and 5′-GpC-3′ sequence contexts and might, in turn, account for the sequence selectivity of the interstrand cross-link formation induced by the native enal-derived 1,N2-dG adducts.

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