Chemical–Biological Fingerprinting: Probing the Properties of DNA Lesions Formed by Peroxynitrite

Abstract

DNA-damaging agents usually produce a vast collection of lesions within the genome. Analysis of these lesions from the structural and biological viewpoints is often complicated by the reality that some of the lesions are chemically fragile, leading to an even larger set of secondary and tertiary products. In an effort to deconvolute complex DNA-damage spectra, a strategy is presented whereby an oligonucleotide containing a specific target for chemical reaction is allowed to react with a DNAdamaging agent. A large collection of HPLC-resolvable modified oligonucleotides is generated, and chromatographically distinct members of the set are then individually characterized using chemical, spectroscopic, biochemical, and genetic probes. The biological component of this "chemicalbiological fingerprinting" tool is the use of polymerase bypass in vivo in cells having defined replication status and quantitative and qualitative patterns of lesion-directed mutagenesis, as key properties that complement physical analysis of modified DNA. This approach was applied to the complex product spectrum generated by peroxynitrite in the presence of CO 2 ; peroxynitrite is a powerful oxidizing and nitrating agent generated as part of immune response. An oligonucleotide containing the primary oxidation product, 7,8-dihydro-8-oxoguanine (8-oxoGua), which is highly susceptible to further oxidation and/or nitration, was treated with peroxynitrite. Using mass spectrometry, coelution with authentic standards, sensitivity to piperidine, recognition and strand cleavage by the DNA repair enzyme MutM, and mutagenicity and genotoxicity in vivo, a matrix was created that defined the properties of the secondary DNA lesions formed when 3-morpholinosydnonimine (SIN-1) delivered a low, constant flux of peroxynitrite to an oligonucleotide containing 8-oxoGua. Two lesions were identified as the diastereomers of spiroiminodihydantoin (Sp), which had been observed previously in nucleoside-based experiments employing SIN-1. A third lesion, triazine, was tentatively identified. However, in addition to these lesions, a number of secondary lesions were generated that had chemical-biological fingerprints inconsistent with that of any known 8-oxoGua-derived lesion described to date. In vitro experiments showed that while some of these newly characterized secondary lesions were removed from DNA by MutM, others were in fact very poor substrates for this repair enzyme. These 8-oxoGua-derived lesions also showed varying degrees of sensitivity to piperidine. Furthermore, all of the secondary lesions observed in this work were potently mutagenic and genotoxic in Escherichia coli. Therefore, while 8-oxoGua itself is nontoxic and only mildly mutagenic in repair-proficient cells, peroxynitrite reveals the promutagenic potential and triggers the covert nature of this DNA lesion. Supporting Information Available: HPLC chromatogram for the reaction between the 8-oxoGua oligonucleotide and SIN-1 (33.3-fold excess of SIN-1), PAGE results following piperidine and Mut...

Formation and processing of DNA damage substrates for the hNEIL enzymes

“…Lastly, a templating Z lesion when bypassed by a polymerase allows insertion of mainly dATP leading to G→T transversion mutations [161]. Cellular studies using the REAP assay verified the in vitro results in which Z preferentially induces G→T transversions ~95% of the time [159]. Besides OG, the other G-derived lesions are nearly ~100% mutagenic causing G→T or G→C transversion mutations.…”

“…Studies to inspect Sp and Gh mutational profiles by the REAP assay verified their ability to cause transversion mutations in ~100% yield [156, 159, 160]. The results indicated that the Sp base stereochemistry impacted the ratio of G→T and G→C transversions.…”

Biologically relevant oxidants and terminology, classification and nomenclature of oxidatively generated damage to nucleobases and 2-deoxyribose in nucleic acids

“…In addition to highly labile normal and all oxidized abasic sites [113], this analysis reveals several oxidatively generated base modifications, which are at least partly con-vertedintoSSBs,suchas2,5-diamino-4 H -imidazolone (Iz) [116], 2,2,4-triamino-5-(2 H )-oxazolone (Oz) [117], 5,6-dihydroxy-5,6-dihydrothymine (ThyGly) [118], FapyAde and, to lesser extent, FapyGua [119]. In contrast to previous claims, 8-oxoGua has been found to be stable under conditions of hot piperidine treatment [117,120]. The strongly alkaline conditions (0.3M NaOH, pH > 13) that are used for the measurement of DNA strand breaks using either the alkaline version of the comet assay or the alkaline eluting technique are incompatible with the instability at high pH of abasic sites, Iz and Oz.…”

Reconciliation of Chemical, Enzymatic, Spectroscopic and Computational Data To Assign the Absolute Configuration of the DNA Base Lesion Spiroiminodihydantoin

“…27 In vivo studies demonstrated that both hydantoins show 100% mutagenesis causing dG→dT and dG→dC (2′-deoxycytidine) transversion mutations if unrepaired. 28–30 These biological observations have implicated the hydantoins as potential culprits for causing mutations that result from oxidative and inflammatory stress leading to disease progression.…”