SOLUTION CONFORMATION OF [AF]DG OPPOSITE A -2 DELETION SITE IN A DNA DUPLEX - INTERCALATION OF THE COVALENTLY ATTACHED AMINOFLUORENE RING INTO THE HELIX WITH BASE DISPLACEMENT OF THE C-8-MODIFIED SYN GUANINE AND ADJACENT UNPAIRED 3'-ADENINE INTO THE MAJOR GROOVE

This paper reports the solution conformation of the covalent aminofluo rene-C-8-deoxyguanosine [AF]dG adduct positioned opposite a -2 deletio n site in a DNA oligomer duplex. The combined NMR and molecular mechan ics computational studies were undertaken on the [AF]dG adduct embedde d in the d(C5-[AF]G6-A7-C8). d(G17-G18) sequence context in a duplex c ontaining 12 residues on the modified strand and 10 on the partner str and, with no bases opposite the [AF]dG6-dA7 segment. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nuclei c acid were assigned following analysis of two-dimensional NMR data se ts in H2O and D2O solution. The solution conformation of the [AF]dG . 2del 12-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanic s computations in torsion angle space. The aminofluorene ring of [AF]d G6 is intercalated between intact Waston-Crick dC5 . dG18 and dC8 . dG 17 base pairs with the deoxyguanosine base of [AF]dG6 in a syn alignme nt displaced into the major groove. The syn glycosidic torsion angle a t [AF]dG6 is supported by both carbon and proton chemical shift data f or the sugar resonances of the modified deoxyguanosine residue. The un paired dA7 base is also looped out of the helix into the major groove with the purine rings of [AF]dG6 and dA7 stacking on each other in the groove. The long axis of the intercalated aminofluorene ring is paral lel to the long axis of the flanking dG . dC base pairs. The intercala tion site is wedge shaped with a pronounced propeller-twisting and buc kling of the dC5 . dG18 base pair. The deoxyguanosine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative base displacement structure of the [AF]d G . 2del 12-mer duplex exhibits several unusually shifted proton reson ances that can be readily accounted for by the ring current contributi ons of the deoxyguanosine purine and carcinogen fluorene aromatic ring s of the [AF]dG6 adduct. We note similarities between the present conf ormation of [AF]dG positioned opposite a -2 deletion site with our ear lier conformational studies of [AF]dG positioned opposite a -1 deletio n site [Mao, B., Cosman, M., Hingerty, B.E., Broyde, S., & Patel, D.J. (1995) Biochemistry 34, 6226-6238]. For both conformations, the amino fluorene carcinogen inserts into the helix at the deletion site throug h base displacement of the modified deoxyguanosine in a syn alignment into the major groove and directed toward its 5'-neighbor in the seque nce. These structures provide a molecular explanation of how transient strand slippage of the lesion-containing segment can be accommodated by a double helix following translesion synthesis.