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

Abstract

Conformations of (R)-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 refined from 1H NMR data. These were the R(61,2) and R(61,3) adducts. Chemical shift perturbations were in the 5'-direction from the sites of adduction; large changes were observed for C5 H5 and H6 in the R(61,2) adduct. The styrene moieties were only partially defined by NOE data. Spectral overlap, particularly for the R(61,2) adduct, prevented complete assignments of the aromatic resonances; likewise, there were insufficient data to orient the CH2OH moieties. Ring flips were slow on the NMR time scale. For the R(61,2) adduct 260 restraints were obtained from NOE data at three mixing times using relaxation matrix analysis; for the R(61,3) adduct 230 restraints were obtained. Structures emergent from molecular dynamics/simulated annealing for the R(61,2) adduct converged to average and maximum pairwise rms differences of 1.3 and 1.7 A, respectively, while those for the R(61,3) adduct converged to average and maximum pairwise rms differences of 1.2 and 1.6 A. Sixth root residual indices of 7.5 and 6.8 x 10(-2) were measured between the refined structures and NOE intensities using relaxation matrix calculations for the R(61,2) and R(61,3) adducts, respectively. The styrene rings were in the 5'-direction from the lesion sites in the major groove. The preferred orientation calculated for the R(61,2) adduct placed the styrene ring edgewise and approximately orthogonal to C5, while that calculated for the R(61,3) duplex had the styrene ring approximately orthogonal to the major groove edges of base pairs A6.T17 and R-SOA7.T16.

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).…”

“…The styryl ring in R-SO points toward the 5Ј-end of the template (i.e. 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).…”

“…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].…”