Wobble dC·dA Pairing 5' to the Cationic Guanine N7 8,9-Dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B_1 Adduct: Implications for Nontargeted AFB_1 Mutagenesis

摘要

The structure of 5'-d(ACATC~(AFB)GATCT)-3'·5'-d(AGATcAATGT)-3', containing the C~5·A~(16) mismatch at the base pair 5' to the modified ~(AFB)G~6, was determined by MR. The characteristic 5'-intercalation of the AFB_1 moiety was maintained. The mismatched C~5·A~(16) pair existed in the wobble conformation, with the C~5 imino nitrogen hydrogen bonded to the A~(16) exocyclic amino group. The wobble pair existed as a mixture of protonated and nonprotonated species. The pK_a for protonation at the A~(16) imino nitrogen was similar to that of the C~5·A~(16) wobble pair in the corresponding duplex not adducted with AFB_1. Overall, the presence of AFB_1 did not interfere with wobble pair formation at the mismatched site. Molecular dynamics calculations restrained by distances derived from NOE data and torsion angles derived from ~1H~3J couplings were carried out for both the protonated and nonprotonated wobble pairs at C~5·A~(16). Both sets of calculations predicted the A~(16) amino group was within 3 A of the C~5 imino nitrogen. The calculations suggested that protonation of the C~5·A~(16) wobbl pair should shift C_5 toward the major groove and shift A~(16) toward the minor groove. The NMR data showed evidence for the presence of a minor conformation characterized by unusual NOEs between T~4 and ~(AFB)G~6. T~4 is two nucleotides in the 5'-direction from the modified base. These NOEs suggested that in the minor conformation nucleotide T~4 was in close proximity to ~(AFB)G~6 than would be expected for duplex DNA. Modeling studies examined the possibility that T~4 transiently paired with the mismatched A~(16), allowing it to come within NOE distance of ~(AFB)G~6. This model structure was consistent with the unusual NOEs associated with the minor conformation. The structural studies are discussed in relationship to nontargeted C->T transitions observed 5' to the modified ~(AFB)G in site-specific mutagenesis experiments [Bailey, E. A., Iyer, R. S., Stone, M. P., Harris, T. M., and Essigmann J. M. (1996) Proc. natl. Acad. Sci. U.S.A. 93, 1535-1539].