Solution structure of a nitrous acid induced DNA interstrand cross-link.

Nitrous acid is a mutagenic agent. It can induce interstrand cross-links in duplex DNA, preferentially at d(CpG) steps: two guanines on opposite strands are linked via a single shared exocyclic imino group. Recent synthetic advances have led to the production of large quantities of such structurally homogenous cross-linked duplex DNA. Here we present the high resolution solution structure of the cross-linked dodecamer [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined), determined by 2D NMR spectroscopy, distance geometry, restrained molecular dynamics and iterative NOE refinement. The cross-linked guanines form a nearly planar covalently linked 'G:G base pair' with only minor propeller twisting, while the cytidine bases of their normal base pairing partners have been flipped out of the helix and adopt well defined extrahelical positions in the minor groove. On the 5'-side of the cross-link, the minor groove is widened to accommodate these extrahelical bases, and the major groove becomes quite narrow at the cross-link. The cross-linked 'G:G base pair' is well stacked on the spatially adjacent C:G base pairs, particularly on the 3'-side guanines. In addition to providing the first structure of a nitrous acid cross-link in DNA, these studies could be of major importance to the understanding of the mechanisms of nitrous acid cross-linking and mutagenicity, as well as the mechanisms responsible for its repair in intracellular environments. It is also the shortest DNA cross-link structure to be described.

Sequence context effect on the structure of nitrous acid induced DNA interstrand cross-links.

In the preceding paper in this journal, we described the solution structure of the nitrous acid cross-linked dodecamer duplex [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined). The structure revealed that the cross-linked guanines form a nearly planar covalently linked 'G:G base pair', with the complementary partner cytidines flipped out of the helix. Here we explore the flanking sequence context effect on the structure of nitrous acid cross-links in [d(CG)]2 and the factors allowing the extrahelical cytidines to adopt such fixed positions in the minor groove. We have used NMR spectroscopy to determine the solution structure of a second cross-linked dodecamer duplex, [d(CGCTACGTAGCG)]2, which shows that the identity of the flanking base pairs significantly alters the stacking patterns and phosphate backbone conformations. The cross-linked guanines are now stacked well on adenines preceding the extrahelical cytidines, illustrating the importance of purine- purine base stacking. Observation of an imino proton resonance at 15.6 p.p.m. provides evidence for hydrogen bonding between the two cross-linked guanines. Preliminary structural studies on the cross-linked duplex [d(CGCGACGTCGCG)]2 show that the extrahelical cytidines are very mobile in this sequence context. We suggest that favorable van der Waals interactions between the cytidine and the adenine 2 bp away from the cross-link localize the cytidines in the previous cross-linked structures.

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment.

Internal motions of d-ribose selectively 2H-labeled at the 2' position were measured using solid state 2H NMR experiments. A sample of d-ribose-2'-d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of -74 and -60 degrees C the C2'-H2' bond was observed to undergo two kinds of motions which were similar to those of C2'-H2'/H2" found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609-8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6+/-0.7 kcal mol(-1), and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy > or =4 kcal mol(-1). At -74 degrees C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at -60 degrees C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2'-H2' bond are most likely from interconversions between C2'-endo and C3'-endo by way of the O4'-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2'-H2' bond within the C2'-endo and C3'-endo potential energy minima.