Flexible 5-Guanidino-4-nitroimidazole DNA Lesions: Structures and Thermodynamics

Reactive oxygen and nitrogen species are products of normal cellular metabolism. In some cases, they are produced specifically to serve essential biological functions, in regulating circulation, energy metabolism, and apoptosis, and they constitute a major defense against pathogens (1, 2). However, these highly reactive species also have the capability of damaging DNA. Base lesions are prominent among the resulting forms of DNA damage (3-9). If not removed by repair enzymes, the processing of the damaged DNA by polymerases may cause mutations, which may in turn initiate cancer (10-14). In addition, aging (15-19), and a variety of diseases such as Alzheimer's disease and cardiovascular disease have been linked to DNA damage caused by oxidative mechanisms (20, 21). Guanine has the lowest redox potential of the four DNA nucleobases and hence is a primary target of oxidative modification (8, 22). 8-oxo-7,8-dihydroguanine (8-oxoG) (23) is considered to be the most important among over 100 oxidized DNA lesions (12, 24, 25). Other oxidation products of guanine include 5-guanidino-4-nitroimidazole (NI), cyanuric acid (Ca), oxaluric acid (Oa), oxazolone (Oz), imidazolone (Iz), urea (Ua), spiroiminodihydantoin (Sp), and guanidinohydantoin (Gh) (26-32). 5-guanidino-4-nitroimidazole (NI), is derived from guanine oxidation by peroxynitrite anion (ONOO−) (30), or reaction of the nitrogen dioxide radical (·NO2) with guanine radicals in DNA (7, 33). These endogenous active oxidative agents are produced by reactive oxygen and nitrogen species in mitochondria and macrophage/inflammatory cells (12). The chemical change reflects nitration, hydrolytic opening of the 6-membered ring of guanine, and decarboxylation (34). The product contains a guanidino group and a nitroimidazole ring. This DNA lesion lacks a Watson-Crick hydrogen bonding edge, and is characterized by opportunities for torsional flexibility (Figure 1A). Of particular importance are the torsion angles δ (N2-C2-N3-C4) and θ (C2-N3-C4-N9) involving the guanidino group. The rotation of θ governs the co-planarity between the guanidino group and the imidazole ring. The planarity of the guanidino group itself is governed primarily by rotation of δ; rotation between C2 and N2 is also feasible. However, all bond lengths, bond angles and dihedral angles are unrestrained in the QM and MD simulations. The opportunities for conformational flexibility can permit various conformations of the lesion. The lesion is notable for its multiple and unique hydrogen bond donor and acceptor groups. These properties have the potential to produce novel structural features in the lesion-containing DNA, which are of interest in relation to its biological function. Figure 1 (A) Structure of 5-guanidino-4-nitroimidazole deoxyribonucleoside. Atom numbers and torsion angles are defined as follows: Glycosidic torsion angle χ is O4′-C1-N9-C4 (68) , δ is N2-C2-N3-C4, and θ is C2-N3-C4-N9. The guanidino ... It has been demonstrated that the NI lesion blocks polymerases and cause mutations, both in vitro and in vivo (29, 34). In vitro primer extension studies show that NI presents a significant replication block in the case of calf thymus pol α and Escherichia coli pol I (Klenow fragment, exo−), but not in the case of human pol β (34). The normal partner C is primarily incorporated opposite the NI lesion in the case of pol β and pol I, while A and G are also inserted in the case of pol I. In the case of pol α, A and G are chiefly incorporated opposite NI (34). In E. coli, the bypass efficiency of the NI lesion is 7.0 ± 1.6% under normal conditions and 57 ± 1% under SOS-induced conditions where bypass polymerases are likely involved. The order of incorporation preference is C > A ≈ T > G under both conditions (29). Oxidative DNA base lesions are repaired by the base excision repair (BER) system (35-37). However, a repair pathway has not yet been identified for the NI lesion. Neither E. coli formamidopyrimidine glycosylase (Fpg) nor endonuclease III (Nth) appear to be effective in NI repair (34). To investigate the biological effects of the NI lesion, a knowledge of the structure of NI itself as well as of NI-damaged DNA duplexes is needed. However, at present, such structural information is not available. We have carried out computational investigations at the levels of the damaged base, nucleoside, and in duplex DNA to elucidate the NI structural preferences. At the base level, quantum mechanical studies provided a geometry-optimized lowest energy planar structure of NI. At the nucleoside level, geometry-optimization reveals the NI base is no longer planar due to steric hindrance to the sugar, consistent with a previous semi-empirical MO calculation (38). At the DNA duplex level, we investigated the NI lesion paired with all four partner bases in a B-form 11mer. Molecular dynamics (MD) simulations in aqueous solution were carried out to obtain ensembles of structures, and trajectories were employed to analyze the structures and to compute free energies. The structural and thermodynamic analyses suggest that the non-planar NI lesion can adopt both syn and anti conformations with the specific preference depending on the partner base, and the guanidino group is positioned in the DNA major and minor grooves, respectively. The unique hydrogen bond properties and the opportunity for flexibility play an important role in the distinct structural features of this unusual lesion.