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

Abstract

Abstract5-Guanidino-4-nitroimidazole (NI), derived from guanine oxidation by reactive oxygen and nitrogen species, contains an unusual flexible ring opened structure, with nitro and guanidino groups which possess multiple hydrogen bonding capabilities. In vitro primer extension experiments with bacterial and mammalian polymerases show that NI incorporates C as well as A and G opposite the lesion, depending on the polymerase. In order to elucidate structural and thermodynamic properties of the mutagenic NI lesion, we have investigated the structure of the modified base itself and the NIcontaining nucleoside with high level quantum mechanical calculations, and have employed molecular modeling and molecular dynamics simulations in solution for the lesion in B-DNA duplexes with four partner bases opposite the NI. Our results show that the NI adopts a planar structure at the damaged-base level. However, in the nucleoside and in DNA duplexes, steric hindrance between the guanidino group and its linked sugar causes NI to be non-planar. The NI lesion can adopt both syn and anti conformations on the DNA duplex level, with the guanidino group positioned in the DNA major and minor grooves, respectively; the specific preference depends on the partner base. Based on hydrogen bonding and stacking interactions, groove dimensions, and bending, we find that the least distorted NI modified duplex contains partner C, consistent with incorporation of C opposite NI. However, hydrogen bonding interactions between NI with partner G or A are also found, which would be compatible with the observed mismatches.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)(16)(17)(18)(19), and a variety † This research is supported by NIH Grant CA-75449 to S. B. and NIH Grant ES-11589 to V. S. and N. E. G. Figure S1 shows representative starting models for the MD simulations. Figure S2 shows stereo views of geometry-optimized NI nucleosides. Figure S3 shows RMSD vs time plots of each molecular dynamics simulation. Figures S4 and S5 show stereo views of the central 5-mer of the NI trajectory-average duplex structures and hydrogen bonds between NI and partner base of high energy structures. Figures S6-S8 show the average bend angles, groove dimensions of the NI damaged DNA, and trajectory plots of torsion angles of the NI lesion, respectively. Tables S1-S5 show added force field parameters, torsion angles, simulation box sizes, and numbers of added waters of initial models, and time windows chosen ...

Base and Nucleotide Excision Repair of Oxidatively Generated Guanine Lesions in DNA

“…1). Molecular modeling studies indicate that the NIm lesions adopt flexible and multiple ring-opened structures with the nitro and guanidino groups providing multiple hydrogen bonding possibilities (26). By contrast, Gh can exist in the forms of two slowly interconverting R and S stereoisomers (81,82).…”

“…On the other hand, the stereoisomeric Sp structures exist as stable and identifiable R and S stereoisomers (24,83). All three lesions, NIm, Gh, and Sp, cause significant thermodynamic destabilization of DNA duplexes (21,25,26,(82)(83)(84)(85)(86)(87), which is a common feature of DNA substrates that are recognized by the NER machinery. Thus, the Sp and Gh lesions are recognized by the prokaryotic UvrABC nuclease (40) as well as by the eukaryotic NER machinery in human cell extracts as demonstrated here.…”

“…Thus, the Sp and Gh lesions are recognized by the prokaryotic UvrABC nuclease (40) as well as by the eukaryotic NER machinery in human cell extracts as demonstrated here. However, the NER resistance of NIm appears to be correlated not with a destabilization but with its conformational flexibility in double-stranded DNA (26). It is noteworthy that the behavior of NIm lesions relative to Sp and Gh lesions is also quite different in translesion bypass experiments catalyzed by the human polymerase ␤ (pol ␤); the NIm lesions are efficiently bypassed by pol ␤ (16), whereas pol ␤ is completely stalled by Sp and Gh lesions (88).…”

“…NMR structural studies in solution indicate that both stereoisomerically distinct Sp lesions (24) perturb adjacent base pairs and thus thermodynamically destabilize oligonucleotide duplexes (25). Molecular modeling studies indicate that the NIm lesions adopt flexible and multiple ringopened structures with the nitro and guanidino groups, providing multiple hydrogen bonding possibilities (26).The Gh and Sp lesions are highly mutagenic, resulting in G 3 C and G 3 T transversion mutations (27). The accumulation of Sp lesions was detected in Nei-deficient Escherichia coli after treatment of these cells with chromate (28) and were also detected in both the liver and colon tissues of Rag2 Ϫ/Ϫ mice at levels ϳ100 times lower than those of 8-oxoG (29).…”

“…NMR structural studies in solution indicate that both stereoisomerically distinct Sp lesions (24) perturb adjacent base pairs and thus thermodynamically destabilize oligonucleotide duplexes (25). Molecular modeling studies indicate that the NIm lesions adopt flexible and multiple ringopened structures with the nitro and guanidino groups, providing multiple hydrogen bonding possibilities (26).…”