Sequence and Stereospecific Consequences of Major Groove α-(N6-Adenyl)-Styrene Oxide Adducts in an Oligodeoxynucleotide Containing the HumanN-rasCodon 61 Sequence

Abstract

The oligodeoxynucleotide d(CGGACAAGAAG):d(CTTCTTGTCCG), containing N-ras codon 61 (underlined), provided a model with which to examine the respective roles of adduct stereochemistry and DNA sequence in modulating the conformations of site-specific diastereomeric α-(N6-adenyl)–styrene oxide adducts. Solution structures of the (R)- and (S)-α-(N6-adenyl)–styrene oxide adducts incorporated at A6 and A7 in this sequence were refined from 1H NMR data. Distance restraints calculated from NOE data using relaxation matrix analysis were incorporated as effective potentials into the total energy equation. Solution structures refined using restrained molecular dynamics which incorporated a simulated annealing protocol were evaluated by complete relaxation matrix methods. Each of these adducts was located in the major groove. Their solution conformations were depen-dent upon both the stereochemistry of adduction and the DNA sequence. Stereochemistry determined the orientation of the styrenyl moieties relative to the lesion sites. For the R diastereomers, the styrene rings were oriented toward the 5′-direction from the sites of adduction. For the S stereoisomers, the styrene rings were oriented toward the 3′-direction from the sites of adduction. Both large and small DNA sequence effects were observed. For the two R adducts, small DNA sequence effects were observed. For the two S adducts, a large sequence effect, in which the S(61, 2) adduct equilibrated between two conformers while the S(61, 3) adduct exhibited only a single conformation, was observed. Potential energy minimization of the S(61, 2) adduct major conformation yielded a structure in which the styrene ring was oriented in the 3′-direction, and interacted primarily with the complementary strand. The minor conformation of the S(61, 2) adduct was not identified. These results are discussed in the context of mutagenesis experiments designed to probe for α-styrene oxide-induced mutations within these same sequences which revealed stereo- and sequence-specific effects in the processing of these lesions. This series of experiments represents the first reported example of ‘walking’ a set of structurally defined adducts along the DNA duplex, with analysis of adduct conformation at each isomeric position on the helix.