Structural Characterization of a (+)-trans-anti-Benzo[a]pyrene-DNA Adduct using NMR, Restrained Energy Minimization, and Molecular Dynamics

Abstract

The (+)-trans-anti-benzo[a]pyrene adduct formed at the N2 amino group of guanine is the major adduct found after metabolic activation of the ubiquitous carcinogen benzo[a]pyrene. The carcinogenic and mutagenic properties of the (+)-trans-anti-BP adduct, as well as related adducts, have been extensively studied. A DNA duplex containing a (+)-trans-anti-benzo[a]pyrene adduct covalently attached to the G8 nucleotide in the sequence d(CCTATGT[BP-G]CAC).d(GTGCACATAGG) was synthesized and the structure characterized by one- and two-dimensional NMR spectroscopy, in conjunction with energy minimization and molecular dynamics. This BP-11-mer duplex exhibits NOESY cross-peaks between benzo[a]pyrene protons and BP-G8, C9, A16, and C17 nucleotide protons that clearly delineate the location of the BP moiety in the minor groove of a B-type duplex with the pyrene ring oriented toward the 5' end of the modified strand. Large upfield shifts of A16 and C17 sugar resonances in the partner strand show that the pyrene moiety is situated near these sugars. Analysis of the spectra was complicated by the presence of chemical exchange line broadening of protons located near the (...T[BP-G]C...).(...GCA...) adduct site which shows the presence of a minor conformation for this BP-modified duplex in which TA is the 5' neighboring base pair. Distance restraints determined from NOESY spectra recorded at 20 degrees C were used in restrained and unrestrained energy minimization and molecular dynamics simulations to obtain a structure characteristic of the predominant conformation of the BP-11-mer duplex. The important structural features of the BP-11-mer are similar to those reported by Cosman et al. [(1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918] for a (+)-trans-anti-BP adduct at a (...C[BP-G]C...).(...GCG...) sequence in which CG is the 5' neighboring base pair. No evidence of a conformational equilibrium was reported in this duplex, from which we conclude that the presence of a 5' TA base pair plays a role in the conformational equilibrium. Watson-Crick base pairing is retained in the predominant conformer of the (+)-trans-anti-BP modified duplex, which provides a visualization of a structure that could allow faithful replication. The exchange rate could not be slowed sufficiently to allow individual distance parameters to be obtained for the minor conformer.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Pair Conformation-Dependent Excision of Benzo[a]Pyrene Diol Epoxide-Guanine Adducts by Human Nucleotide Excision Repair Enzymes

“…It has been shown that carcinogen-DNA lesions are subject to strong sequence-dependent conformational polymorphisms. For example, in the 5Ј-TCGCT-3Ј sequence context of this study (ϩ)-trans-BPDE-N 2 -dG induces a structural motif characterized by Watson-Crick base pairing (7) but a single nucleotide change on the 5Ј side of the same lesion is associated with the appearance of an additional conformer (13). Similarly, acetylaminofluorene-C 8 -dG adducts have been associated with sequence-specific polymorphisms in the extent of base pair denaturation (15,18,38,45), and these variations constitute an important source of repair heterogeneity (34).…”

Cytosine methylation determines hot spots of DNA damage in the human P 53 gene

“…The structural influence of the cytosine methyl group on B-DNA conformation seems to be insignificant as this group is located in the major groove of the DNA helix (48). In contrast, the B[a]P ring of (ϩ)-trans adducts is positioned in the minor groove and is directed toward the 5Ј end of the modified strand (49,50). BPDE forms noncovalent intercalative complexes with double-stranded DNA that are thought to precede covalent binding of BPDE to DNA (51).…”

Dynamics of a Benzo[a]pyrene-derived Guanine DNA Lesion in TGT and CGC Sequence Contexts: Enhanced Mobility in TGT Explains Conformational Heterogeneity, Flexible Bending, and Greater Susceptibility to Nucleotide Excision Repair

“…29 In the TG*C sequence context, the dominant conformation is of the same MG type, but a minor conformation may involve insertion of the BP moiety into the duplex with disruption of Watson-Crick pairing at the lesion site. 30 More subtle sequence effects on the MG conformations of the 10S (+)-transanti- [BP]-N 2 -dG adduct in 5′-CG*G and 5′-GG*C sequence contexts have been recently reported 31 and have been attributed to the effects of steric hindrance between the exocyclic amino groups of guanines and the BP residues that compete for the same space in the MG. Interestingly, for the same 10S adduct in a CG*C sequence context in an 11/10-mer duplex in which the G* had no partner, a base-displaced/intercalated (BD) conformation was adopted in which the BP rings were inserted into the helix with the modified guanine residue, as well as the puckered benzylic ring positioned on the major groove side of the helix. 32 The CG*C and TG*T sequences are of unusual interest because the nature of the flanking bases affects not only the adduct conformations in double-stranded DNA, but also a number of other properties.…”