AN INTERCALATED AND THERMALLY STABLE FAPY ADDUCT OF AFLATOXIN B-1 IN A DNA DUPLEX - STRUCTURAL REFINEMENT FROM H-1-NMR

The structure of a formamidopyrimidine (FAPY) adduct arising from imid azole ring opening of the initially formed trans-8,9-dihydro-8-(N7-gua nyl)-9-hydroxyaflatoxin B-1 adduct under basic conditions and position ed in the 5'-d(CTAT(FAPY)GATTCA)-3' . 5'-d(TGAATCATAG)-3' oligodeoxynu cleotide was determined. The FAPY adduct may be a major progenitor of aflatoxin B-1-induced mutations in DNA. The freshly prepared sample sh owed biphasic melting, with transitions at 28 and 56 degrees C. NMR in itially showed multiple subspectra. Over a period of several days at 4 degrees C, the sample converted to a single species with a T-m of 56 degrees C, 15 degrees C greater than the unmodified duplex. The deoxyr ibose was in the beta configuration about the anomeric carbon, evidenc ed by NOEs between (FAPY)G(5) H3', H2', H2'', and H1'. FAPY formation resulted in the loss of the guanine H8 proton, and the introduction of the formyl proton, which showed NOEs to (FAPY)G(5) H1' and A(6) N6H(a ). A total of 31 NOEs from AFB(1) to DNA protons were observed, mostly to the 5'-neighboring base, T-4 in the modified strand. Sequential NO Es were interrupted between T-4 and (FAPY)G(5) in the modified strand, between C-16 and A(17) in the complementary strand, and between T-4 N 3H and (FAPY)G(5) N1H. An NOE between (FAPY)G(5) N1H and C-16 N4H show ed intact hydrogen bonding at (FAPY)G(5) . C-16. Upfield chemical shif ts were observed for T-4 H6 and A(17) H8. Molecular dynamics calculati ons converged with pairwise rmsd differences of <0.9 Angstrom. The six th root residual was 8.7 x 10(-2). The AFB(1) moiety intercalated from the major groove between (FAPY)G(5) and T-4 . A(17) and stacked with T-4 and (FAPY)G(5) and partially stacked with A(17). The base step bet ween T-4 A(17) and (FAPY)G(5) . C-16 was increased from 3.4 to 7 Angst rom. The duplex unwound by about 15 degrees. The FAPY formyl group was positioned to form a hydrogen bond with A(6) N6H(a). Strong stacking involving the AFB(1) moiety, and this hydrogen bond explains the therm al stabilization of four base pairs by this adduct, and may be a signi ficant factor in its processing.