The A and B conformations of DNA and RNA subunits. Potential energy calculations for dGpdC

In order to obtain a molecular picture of the A and B forms of a DNA subunit, potential energy calculations have been made for dGpdC with C(3′)-endo and C(2′)-endo [or C(3′)-exo] sugar puckerings. These are compared with results for GpC. The global minima for dGpdC and GpC are almost identical. They are like A-form duplex DNA and RNA, respectively, with bases anti, the ω′, ω angle pair near 300°, 280°, and sugar pucker C(3′)-endo. For dGpdC, a B-form helical conformer, with sugar pucker C(2′)-endo and ω′ = 257°, ω = 298°, is found only 0.4 kcal/mol above the global minimum. A second low-energy conformation (2.3 kcal/mol) has ω′ = 263°, ω = 158° and ψ near 180°. This has dihedral angles like the original Watson–Crick model of the double helix. In contrast, for GpC, the C(2′)-endo B form is 6.9 kcal/mol above the global minimum. These theoretical results are consistent with experimental studies on DNA and RNA fibers. DNA fibers exist in both A and B forms, while RNA fibers generally assume only the A form. A low-energy conformation unlike the A or B forms was found for both dGpdC and GpC when the sugars were C(3′)-endo. This conformation—ω′,ω near 20°,80°—was not observed for C(2′)-endo dGpdC. Energy surface maps in the ω′,ω plane showed that C(2′)-endo dGpdC has one low-energy valley. It is in the B-form helical region (ω′ ～ 260°, ω ～ 300). When the sugar pucker is C(3′)-endo, dGpdC has two low-energy regions: the A-form helical region and the region with the minimum at ω′ = 16°, ω = 85°.     

Simulating structural and thermodynamic properties of carcinogen-damaged DNA

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.     

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability

A key issue in the nucleotide excision repair (NER) of bulky carcinogen–DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phe nanthrene and the corresponding (–)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts. Duplexes containing the 1R (+) or 1S (–) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (–)-trans-anti-B[a]P-N⁶-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson– Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson–Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.     

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.     

A bulky DNA lesion derived from a highly potent polycyclic aromatic tumorigen stabilizes nucleosome core particle structure

The impact of a bulky DNA lesion on the structure and dynamics of a nucleosome core particle (NCP) containing a lesion derived from the unusually potent tumorigen dibenzo[a,l]pyrene that resists nucleotide excision repair (NER) in free DNA was investigated using 65 ns molecular dynamics simulations. Our results reveal that, relative to unmodified NCP, the lesion stabilizes the nucleosome via stacking interactions, improved Watson-Crick base pairing, hydrogen bonding between DNA and histones, and damped dynamics. These findings suggest that such lesions should be as resistant to NER in the nucleosome environment as they are in free DNA.     

The human DNA repair factor XPC-HR23B distinguishes stereoisomeric benzo[a]pyrenyl-DNA lesions

Benzo[a]pyrene (B[a]P), a known environmental pollutant and tobacco smoke carcinogen, is metabolically activated to highly tumorigenic B[a]P diol epoxide derivatives that predominantly form N(2)-guanine adducts in cellular DNA. Although nucleotide excision repair (NER) is an important cellular defense mechanism, the molecular basis of recognition of these bulky lesions is poorly understood. In order to investigate the effects of DNA adduct structure on NER, three stereoisomeric and conformationally different B[a]P-N(2)-dG lesions were site specifically incorporated into identical 135-mer duplexes and their response to purified NER factors was investigated. Using a permanganate footprinting assay, the NER lesion recognition factor XPC/HR23B exhibits, in each case, remarkably different patterns of helix opening that is also markedly distinct in the case of an intra-strand crosslinked cisplatin adduct. The different extents of helix distortions, as well as differences in the overall binding of XPC/HR23B to double-stranded DNA containing either of the three stereoisomeric B[a]P-N(2)-dG lesions, are correlated with dual incisions catalyzed by a reconstituted incision system of six purified NER factors, and by the full NER apparatus in cell-free nuclear extracts.     

Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N²-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5′-···GG··· dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5′-···CG*GC··· and 5′-···CGG*C··· sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 5′ along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 5′-···CGG*C··· case, the 5′-flanking G · C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 5′-···CG*GC··· context, there is no untwisting, but there is significant destabilization of the 5′-flanking Watson–Crick base pair. The minor groove width opens near the lesion in both cases, but more for 5′-···CGG*C···. Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.     

Lesion specificity in the base excision repair enzyme hNeil1: modeling and dynamics studies

Base excision repair (BER) is the major pathway employed to excise oxidized DNA lesions. Human Neil1, a versatile glycosylase in the BER pathway, repairs a diverse array of oxidative lesions; however, the most prevalent, 8-oxo-7,8-dihydroguanine (8-oxoG), is only weakly excised. The structural origin of hNeil1's ability to repair a variety of lesions but not 8-oxoG is a model system for connecting enzyme structure and lesion-recognition specificity. To elucidate structural properties determining hNeil1's substrate specificities, we have investigated it in complex with two pairs of representative well-repaired substrates: the R- and S-spiroiminodihydantoin (Sp) stereoisomers, nonplanar further oxidation products of guanine, and the 5R,6S- and 5S,6R-thymine glycol (Tg) stereoisomers, the most prevalent oxidative lesions of thymine. We also investigate the poorly repaired 8-oxoG. We employed molecular modeling and 10 ns molecular dynamics (MD) simulations. The results of our investigations provide structural explanations for the ability of hNeil1 to excise a variety of oxidative lesions: they possess common chemical features, namely, a pyrimidine-like ring and shared hydrogen bond donor-acceptor properties, which allow the lesions to fit well in the binding pocket, which is somewhat flexible. However, the planar 8-oxoG is not as well accommodated in the shallow and comparatively cramped recognition pocket; it has fewer hydrogen bonding interactions with the enzyme and a solvent exposed six-membered ring, consistent with its poor repair susceptibility by this enzyme.     

Equilenin-derived DNA adducts to cytosine in DNA duplexes: structures and thermodynamics

The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.