Solution structures of aminofluorene [AF]-stacked conformers of the syn [AF]-C-8-dG adduct positioned opposite dC or dA at a template-primer junction

A solution structural study has been undertaken on the aminofluorene-C-8-dG ([AF]dG) adduct located at a single-strand-double-strand d(A1-A2-C3-[AF] G 4-C5-T6-A7-C8-C9-A10-T11-C12-C13) d(G14-G15-A16-T17-G18-G19-T20-A21-G22-N23 ) 13/10-mer junction (N = C or A) using proton-proton distance restraints d erived from NMR data in combination with intensity-based relaxation matrix refinement computations. This single-strand-double-strand junction models o ne arm of a replication fork composed of a 13-mer template strand which con tains the [AF]dG modification site and a 10-mer primer strand which has bee n elongated up to the modified guanine with either its complementary dC par tner or a dA mismatch. The solution structures establish that the duplex se gment retains a minimally perturbed B-DNA conformation with Watson-Crick hy drogen bonding retained up to the dC5.dG22 base pair. The guanine ring of t he [AF]dG4 adduct adopts a syn glycosidic torsion angle and is displaced in to the major groove when positioned opposite dC or dA residues. This base d isplacement of the modified guanine is accompanied by stacking of one face of the aminofluorene ring of [AF]dG4 with the dC5.dG22 base pair, while the other face of the aminofluorene ring is stacked with the purine ring of th e nonadjacent dA2 residue. By contrast, the dC and dA residues opposite the junctional [AF]dG4 adduct site adopt distinctly different alignments. The dC23 residue positioned opposite the adduct site is looped out into the min or groove by the aminofluorene ring. The syn displaced orientation of the m odified dG with stacking of the aminofluorene and the looped out position o f the partner dC could be envisioned to cause polymerase stalling associate d with subsequent misalignment leading to frameshift mutations in appropria te sequences. The dA23 residue positioned opposite the adduct site is posit ioned in the major groove with its purine ring aligned face down over the v an der Waals surface of the major groove and its amino group directed towar d the T6.A21 base pair. The Hoogsteen edge of the modified guanine of [AF]d G4 and the Watson-Crick edge of dA23 positioned opposite it are approximate ly coplanar and directed toward each other but are separated by twice the h ydrogen-bonding distance required for pairing. This structure of [AF]dG opp osite dA at a model template-primer junctional site can be compared with a previous structure of [AF]dG opposite dA within a fully paired duplex [Norm an, D., Abuaf, P., Hingerty, B. E., Live, D., Grunberger, D., Broyde, S., a nd Patel, D. J. (1989) Biochemistry 28, 7462-7476]. The alignment of the Ho ogsteen edge of [AF]dG (syn) positioned opposite the Watson-Crick edge of d A (anti) has been observed for both systems with the separation greater in the case of the junctional alignment in the model template-primer system. H owever, the aminofluorene ring is positioned in the minor groove in the ful ly paired duplex while it stacks over the junctional base pair in the templ ate-primer system. This suggests that the syn [AF]dG opposite dA junctional alignment can be readily incorporated within a duplex by a translation of this entity toward the minor groove.