An Intercalated and Thermally Stable FAPY Adduct of Aflatoxin B₁ in a DNA Duplex:  Structural Refinement from ¹H NMR

Abstract

The structure of a formamidopyrimidine (FAPY) adduct arising from imidazole ring opening of the initially formed trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct under basic conditions and positioned in the 5'-d(CTATFAPYGATTCA)-3'*5'-d(TGAATCATAG)-3' oligodeoxynucleotide was determined. The FAPY adduct may be a major progenitor of aflatoxin B1-induced mutations in DNA. The freshly prepared sample showed biphasic melting, with transitions at 28 and 56 degreesC. NMR initially showed multiple subspectra. Over a period of several days at 4 degreesC, the sample converted to a single species with a Tm of 56 degreesC, 15 degrees C greater than the unmodified duplex. The deoxyribose was in the beta configuration about the anomeric carbon, evidenced by NOEs between FAPYG5 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 FAPYG5 H1' and A6 N6Ha. A total of 31 NOEs from AFB1 to DNA protons were observed, mostly to the 5'-neighboring base, T4 in the modified strand. Sequential NOEs were interrupted between T4 and FAPYG5 in the modified strand, between C16 and A17 in the complementary strand, and between T4 N3H and FAPYG5 N1H. An NOE between FAPYG5 N1H and C16 N4H showed intact hydrogen bonding at FAPYG5*C16. Upfield chemical shifts were observed for T4 H6 and A17 H8. Molecular dynamics calculations converged with pairwise rmsd differences of <0.9 A. The sixth root residual was 8.7 x 10(-2). The AFB1 moiety intercalated from the major groove between FAPYG5 and T4*A17, and stacked with T4 and FAPYG5 and partially stacked with A17. The base step between T4*A17 and FAPYG5*C16 was increased from 3.4 to 7 A. The duplex unwound by about 15 degrees. The FAPY formyl group was positioned to form a hydrogen bond with A6 N6Ha. Strong stacking involving the AFB1 moiety, and this hydrogen bond explains the thermal stabilization of four base pairs by this adduct, and may be a significant factor in its processing.

NEIL1 protects against aflatoxin-induced hepatocellular carcinoma in mice

“…1C) and consequent stabilization of the DNA as measured by increased melting temperature (12). As with many bulky DNA adducts, DNA repair of both AFB 1 -N7-dG and AFB 1 -Fapy-dG occurs via the NER pathway in Escherichia coli (18).…”

“…Therefore, based on the broad substrate range of NEIL1, it was hypothesized that repair of AFB 1 -Fapy-dG could be initiated by NEIL1 as the first step of the BER pathway. (12). (D-F) hNEIL1-catalyzed incision of lesion-containing DNA under single-turnover conditions.…”

“…The high efficiency with which NEIL1 catalyzed the release of the AFB 1 -Fapy-dG adducts was unanticipated because the base modification stabilizes the melting temperature of a 12-mer duplex DNA by 15°C (12), and this increase would make the modified nucleotide resistant to extrahelical flipping as a prerequisite of glycosyl bond scission. Sterically, this adduct also represents the largest DNA base modification reported as a substrate for any DNA glycosylase.…”

“…The AFB 1 -Fapy-dG adduct can exist in two interconvertible anomeric forms, α and β, that differ by orientation of the glycosyl bond relative to the deoxyribose (11). Although the α and β anomers equilibrate at an approximately equal ratio in single-stranded environments, the β form is strongly favored in duplex DNA where it intercalates within DNA (12) (Fig. 1C).…”

Mutational spectra of aflatoxin B 1 in vivo establish biomarkers of exposure for human hepatocellular carcinoma

“…There, the toxin is converted by phase I enzymes, such as cytochrome P450 1A2 and 3A4 (6, 7), into metabolites that include the exo-epoxide, which is the primary chemical precursor to AFB 1 -mediated mutagenic and carcinogenic events. This epoxide intercalates 5′ to guanine targets in DNA before covalent bonding with the guanyl N7 atom (8), ultimately forming a stereospecific AFB 1 -N 7 -Gua adduct (Fig. 1).…”