Direct Synthesis and Characterization of Site-Specific Adenosyl Adducts Derived from the Binding of a 3,4-Dihydroxy-1,2-epoxybenzo[c]phenanthrene Stereoisomer to an 11-mer Oligodeoxyribonucleotide

Abstract

Site-specifically modified oligonucleotides were obtained in milligram quantities by reacting racemic 3t,4r-dihydroxy-1,2t-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE-2, or anti-B[c]PhDE) with the single deoxyadenosine (dA) residue in the oligodeoxynucleotide d(CTCTCACTTCC). Enzyme digestion of the covalently modified oligonucleotides with the exonuclease spleen phosphodiesterase yielded covalently linked B[ca]PhDE-N6-deoxyadenosyl monophosphate (dAMP) adducts. Comparisons of the reverse phase HPLC retention times and CD spectra of these B[c]PhDE-3'-dAMP mononucleotide adducts, with those of standards derived from the reaction of the enantiomers (+)- and (-)-anti-B[c]PhDE with 3'-dAMP, show that two major oligonucleotide adducts (I and II) were obtained upon reacting racemic anti-B[c]PhDE with d(CTCTCACTTCC). In oligonucleotide adduct I, the lesion is a (+)-trans-anti-B[c]PhDE-N6-dA residue, and in oligonucleotide adduct II it is a (-)-trans-anti-B[c]PhDE-N6-dA residue. These assignments were further confirmed using a standard 32P postlabeling assay of B[c]PhDE-3'-dAMP mononucleotide adducts obtained from the digestion of oligonucleotides I and II by spleen phosphodiesterase. The melting points (Tm) of duplexes of modified oligonucleotides I and II and their natural complementary strands are not affected significantly by the presence of the covalently bound benzo[c]phenanthrenyl residues. Opposite stereoselective resistance to enzyme digestion by the exonucleases snake venom phosphodiesterase and spleen phosphodiesterase is exhibited by the stereoisomeric (+)-trans- and (-)-trans-anti-B[c]PhDE-modified oligonucleotide adducts I and II; these results are consistent with the intercalative insertion of the benzo[c]phenanthrenyl residues on the 5'-side of the modified dA residue in adduct I, and its insertion on the 3'-side of the dA residue in adduct II, as observed in the duplexes by high resolution NMR techniques [Cosman et al. (1993) Biochemistry 32, 12488-12497, and Cosman et all, Biochemistry, in press.

Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: relating biological consequences to conformational preferences

“…The BPhDE-modi®ed 11mers used in this work were prepared and puri®ed as reported elsewhere (28). The BPhDEmodi®ed and unmodi®ed oligodeoxynucleotides exhibit different mobilities during electrophoresis, with the bulky carcinogen moiety causing the damaged molecules to migrate more slowly than their undamaged counterparts.…”

“…The 11mer 5¢-CTCTCACTTCC-3¢ with a BPhDE attached to the N 6 position of the sole adenine served as the source of a site-speci®c, stereochemically pure adduct (28). The oligomers used to prepare the template DNA were phosphorylated at the 5¢-end with 10 U of T7 polynucleotide kinase and 1 mM ATP in 10 ml of 70 mM Tris±HCl (pH 7.6), 10 mM MgCl 2 and 5 mM DTT.…”

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

“…30,32 A number of other fjord PAH-N 6 -dA adducts with intercalative adduct conformations share similar features. 33 Neither the fjord B[c]Ph-N 6 -dA adducts in duplex I (Figures 3 and 4) nor several other fjord PAH-dA adducts 7 are excised by human NER enzymes.…”

Synthesis and Characterization of Site-Specific and Stereoisomeric Fjord Dibenzo[a,l]pyrene Diol Epoxide−N6-Adenine Adducts: Unusual Thermal Stabilization of Modified DNA Duplexes

“…An exception to these T m patterns was reported with adducts derived from the binding of the fjord region B[c]-Ph and B[g]C diol epoxides to N 6 -dA positioned sitespecifically in oligonucleotide duplexes. Neither the 1R (+)-trans-anti-B[c]PhDE-N 6 -dA nor the stereoisomeric 1S (-)-trans-anti-B[c]PhDE-N 6 -dA adducts induce any significant changes in T m of an 11-mer oligonucleotide duplex (48). Even more remarkable, in the case of the of the 14R (+)-trans-anti-B[g]CDE-N 6 -dA adduct, the T m was higher than in the case of the unmodified duplex, but the T m of the stereoisomeric 14S adduct was lower (27).…”