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Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties.

ABSTRACT: Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N(6)-(2-Deoxy-?,?-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme involves the synthesis of an oligonucleotide precursor containing a 5-nitropyrimidine moiety at the desired lesion site via standard solid-phase procedures. Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation and subsequent formylation. NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals the presence of rotameric and anomeric species. The latter have been characterized by synthesizing the tridecamer oligodeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol to resolve the isomeric components. Hybridization of the chromatographically isolated fractions with their complementary d(CGCATGCGTACGC) counterpart yields two Fapy-dG·C duplexes that are differentially destabilized relative to the canonical G·C parent. The resultant duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of ?- and ?-anomers, the former more destabilizing than the latter. These anomer-specific impacts are discussed in terms of differential repair enzyme recognition, processing and translesion synthesis.

SUBMITTER: Lukin M

PROVIDER: S-EPMC3141231 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties.

Lukin Mark M Minetti Conceição A S A CA Remeta David P DP Attaluri Sivaprasad S Johnson Francis F Breslauer Kenneth J KJ de Los Santos Carlos C

Nucleic acids research 20110316 13

Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N(6)-(2-Deoxy-α,β-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme invo ...[more]

PMID: 21415012

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Project description:Genetic information, irrespective of cell type (normal or cancerous), is exposed to a range of harmful factors, which can lead to more than 80 different types of DNA damage. Of these, oxoG and FapyG have been identified as the most abundant in normoxic and hypoxic conditions, respectively. This article considers d[AFapyGAOXOGA]*[TCTCT] (oligo-FapyG) with clustered DNA lesions (CDLs) containing both the above types of damage at the M06-2x/6-31++G** level of theory in the condensed phase. Furthermore, the electronic properties of oligo-FapyG were analysed in both equilibrated and non-equilibrated solvation-solute interaction modes. The vertical/adiabatic ionization potential (VIP, AIP) and electron affinity (VEA, AEA) of the investigated ds-oligo were found as follows in [eV]: 5.87/5.39 and -1.41/-2.09, respectively. The optimization of the four ds-DNA spatial geometries revealed that the transFapydG was energetically privileged. Additionally, CDLs were found to have little influence on the ds-oligo structure. Furthermore, for the FapyGC base-pair isolated from the discussed ds-oligo, the ionization potential and electron affinity values were higher than those assigned to OXOGC. Finally, a comparison of the influence of FapyGC and OXOGC on charge transfer revealed that, in contrast to the OXOGC base-pair, which, as expected, acted as a radical cation/anion sink in the oligo-FapyG structure, FapyGC did not significantly affect charge transfer (electron-hole and excess-electron). The results presented below indicate that 7,8-dihydro-8-oxo-2'-deoxyguanosine plays a significant role in charge transfer through ds-DNA containing CDL and indirectly has an influence on the DNA lesion recognition and repair process. In contrast, the electronic properties obtained for 2,6-diamino-4-hydroxy-5-foramido-2'deoxypyrimidine were found to be too weak to compete with OXOG to influence charge transfer through the discussed ds-DNA containing CDL. Because increases in multi-damage site formation are observed during radio- or chemotherapy, understanding their role in the above processes can be crucial for the efficiency and safety of medical cancer treatment.

Project description:Modified nucleotides often hinder and/or decrease the fidelity of DNA polymerases. Tandem lesions, which are comprised of DNA modifications at two contiguous nucleotide positions, can be even more detrimental to genome stability. Recently, tandem lesions containing 5-formyl-2'-deoxyuridine (5fdU) flanked at the 5'-position by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo) or N-(2-deoxy-α,β-D-erythropentofuranosyl)-N-(2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy•dG) were discovered. We examined the replication of 5'- 8-OxodGuo-5fdU and 5'-Fapy•dG-5fdU tandem lesions in HEK 293T cells and several polymerase deficient variants by transfecting single-stranded vectors containing them. The local sequence of the tandem lesions encompasses the 273 codon of the p53 gene, a mutational hot-spot. The bypass efficiency and mutation spectra of the tandem lesions were compared to those of the isolated lesions. Replication of weakly mutagenic 5-fdU is little changed when part of the 5'- 8-OxodGuo-5fdU tandem lesion. G → T transversions attributable to 8-OxodGuo increase > 10-fold when the tandem lesion is bypassed. 5'-Fapy•dG-5fdU has a synergistic effect on the error-prone bypass of both lesions. The mutation frequency (MF) of 5'-Fapy•dG-5fdU increases 3-fold compared to isolated Fapy•dG. In addition, a 5'-adjacent Fapy•dG significantly increases the MF of 5fdU. The major mutation, G → T transversions, decrease by almost a third in hPol κ- cells, which is the opposite effect when isolated Fapy•dG in the same sequence context is replicated in HEK 293T cells in the same sequence. Steady-state kinetics indicate that hPol κ contributes to greater G → T transversions by decreasing the specificity constant for dCTP compared to an isolated Fapy•dG. The greater conformational freedom of Fapy•dG compared to 8-OxodGuo and its unusual ability to epimerize at the anomeric center is believed to be the source of the complex effects of 5'-Fapy•dG-5fdU on replication.

Dataset Information

Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties.

Publications

Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties.

Similar Datasets

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