Major Groove (S)-α-(N⁶-Adenyl)styrene Oxide Adducts in an Oligodeoxynucleotide Containing the Human N-ras Codon 61 Sequence:  Conformations of the S(61,2) and S(61,3) Sequence Isomers from ¹H NMR

Abstract

The (S)-alpha-(N6-adenyl)styrene oxide adducts at positions X6 in d(CGGACXAGAAG). d(CTTCTTGTCCG) and X7 in d(CGGACAXGAAG).d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human n-ras protooncogene, were examined by 1H NMR. These were the S(61,2) and S(61,3) adducts. Chemical shift perturbations were in the 3'-direction from the sites of adduction; upfield changes associated with the styrene aromatic ring current were noted for S-SOA6 H2 and H1', T16 N3H, H6, and CH3 resonances in the S(61,2) oligomer. In the S(61,3) oligomer, S-SOA7 H1', T16 H1', C15 N4Ha, and H5 shifted upfield. The styrene aromatic rings flipped rapidly on the NMR time scale; under these conditions the ortho and meta aromatic protons were equivalent. A 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. For the S(61,3) adduct, 291 restraints were obtained from NOE data at three mixing times using relaxation matrix analysis. The emergent structures refined to an average rms difference of 1.3 A, determined by pairwise analysis. These were compared to NOE intensity data; the calculated sixth root residual index was 9.2 x 10-2 at 250 ms. In the refined structure, the styrene ring was also oriented in the 3'-direction and interacted with the complementary strand. The minor conformation of the S(61,2) adduct was not identified. These results contrasted with the diastereomeric R(61,2) and R(61,3) adducts, which underwent slow ring flips on the NMR time scale and for which small sequence effects involving the minimum energy conformation of the styrene ring were observed.

Vertical-scanning Mutagenesis of a Critical Tryptophan in the “Minor Groove Binding Track” of HIV-1 Reverse Transcriptase

“…The methods for the chemical synthesis of these unique lesions has been described elsewhere (23,24). The solution structure of the R-and S-SO lesions within an oligodeoxynucleotide duplex revealed that the adducts, which lie in the major groove, are well accommodated by the DNA duplex and do not distort the local helix geometry (21,22). However, the R-and S-SO stereoisomers differ in their spatial orientation; the R-SO N 6 -adenine adduct is directed toward the 5Ј-end of the template (21), whereas the S-SO adduct is directed toward the 3Ј-terminus of the template (22).…”

“…The SO lesions used in this study are known to lie in the major groove without causing a significant perturbation to the local DNA structure (21,22). In contrast, DNA binding by RT is predominantly through the DNA minor groove and RT is unable to bypass R-or S-SO lesions situated in the DNA minor groove (34).…”

“…in the direction of polymerization) (21), but the styryl ring of S-SO points toward the 3Ј-end of the template (i.e. facing the oncoming polymerase) (22). Thus, a perturbation in the DNA that is triggered by an S-SO adduct would be expected to be detected by the polymerase before a similar distortion created by an R-SO lesion occupying the same position in the template.…”

“…An earlier study analyzing the effects of site-defined styrene oxide (SO) DNA adducts on RT-mediated DNA synthesis suggested that this monocyclic aromatic hydrocarbon major groove adduct causes RT to terminate polymerization. Since these major groove lesions do not significantly alter the solution structure of DNA (21,22), it was suggested that the adducts may restrict DNA bending in the template-primer stem and/or interfere with key RT-DNA contacts (possibly those supplied by MGBT residues) (23). In this report, we examine whether the major groove SO lesions induce structural alterations in the DNA minor groove that are monitored by residues known to interact in the minor groove in the vicinity of the DNA bend (15,16).…”

“…2. Unadducted templates (U) are in lanes 1,4,7,10,13,16,19,22, and 25; R-SO-containing templates (R) are in lanes 2,5,8,11,14,17,20,23, and 26; S-SO-containing templates (S) are in lanes 3,6,9,12,15,18,21,24, and 27. The amount of polymerase and time of DNA synthesis were as follows: wild-type, 12.5 fmol for 4 min (lanes 1-3); W266F, 12 fmol for 3 min (lanes 4 -6); W266Y, 3 fmol for 3 min (lanes 7-9); W266I, 12 fmol for 3 min (lanes 10 -12); W266L, 12 fmol for 3 min ( lanes 13-15); W266V, 12 fmol for 3 min (lanes 16 -18); W266A, 12.5 fmol for 1.5 min (lanes 19 -21); W266R, 15.5 fmol for 3 min (lanes 22-24); W266E, 3 fmol for 3 min (lanes [25][26][27].…”