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NMR Evidence for Syn-Anti Interconversion of a Trans Opened (10R)-dA Adduct of Benzo[a]pyrene (7S,8R)-Diol (9R,10S)-Epoxide in a DNA Duplex^†

Supporting Info

D. E. Volk,^‡ J. S. Rice,^‡^§ B. A. Luxon,^‡ H. J. C. Yeh, C. Liang, G. Xie, J. M. Sayer, D. M. Jerina, and D. G. Gorenstein*^‡

Sealy Center for Structural Biology and Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1157 and National Institute of Diabetes and Digestive and Kidney Diseases, The National Institutes of Health, Bethesda, Maryland 20892-0820

Biochemistry, 2000, 39 (46), pp 14040–14053

DOI: 10.1021/bi001669l

Publication Date (Web): October 25, 2000

†

This research was supported by NIH (ES06839 and 1P30 ES06676), Welch Foundation (H-1296), and Sealy and Smith Foundation grants to D.G.G. Building funds were provided by the NIH (1CO6CA59098). The project was funded in part by an NIH fellowship to D.E.V. (T32 AI07536).

‡

University of Texas Medical Branch.

Present Address: MD Anderson Cancer Center, Houston, TX 77030-4095.

NIDDK.

Address correspondence to this author. Telephone: 409-747-6800; fax: 409-747-6850; e-mail: david@nmr.utmb.edu.

Abstract

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T₁A₂G₃T₄C₅A₆A₇*G₈G₉G₁₀C₁₁A₁₂)−d(T₁₃G₁₄C₁₅C₁₆C₁₇T₁₈T₁₉G₂₀A₂₁C₂₂T₂₃A₂₄), containing a 10R adduct at dA*₇that corresponds to trans addition of the N⁶-amino group of dA₇ to (−)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(−)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5‘-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S,R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.