Recent studies distinguish the biological and pharmacological effects of nitroxyl (HNO) from its oxidized/deprotonated product nitric oxide (NO), but lack of HNO detection methods limits understanding its in vivo mechanisms and the identification of endogenous sources. We previously demonstrated that reaction of HNO with triarylphosphines provides aza-ylides and HNO-derived amides, which may serve as stable HNO biomarkers. We now report a kinetic analysis for the trapping of HNO by phosphines, ligations of enzyme-generated HNO, and compatibility studies illustrating the selectivity of phosphines for HNO over other physiologically relevant nitrogen oxides. Quantification of HNO using phosphines is demonstrated using an HPLC-based assay and ligations of phosphine carbamates generate HNO-derived ureas. These results further demonstrate the potential of phosphine probes for reliable biological detection and quantification of HNO.
Nucleotide incorporation and extension opposite N 2 -ethylGua by DNA polymerase was measured and structures of the DNA polymerase -N 2 -ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase opposite N 2 -ethyl-Gua was determined by steady state kinetic analysis with Mg 2؉ or Mn 2؉ as the activating metal. DNA polymerase incorporates dCMP opposite N 2 -ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg 2؉ and with greater efficiencies in the presence of Mn 2؉ . However, the fidelity of nucleotide incorporation by DNA polymerase opposite N 2 -ethyl-Gua and Gua using Mn 2؉ is lower relative to that using Mg 2؉ indicating a metal-dependent effect. DNA polymerase extends from the N 2 -ethyl-Gua:Cyt 3 terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase catalyzed reaction is well suited for N 2 -ethyl-Gua bypass. The structure of DNA polymerase with N 2 -ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase complexed with N 2 -ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.2 is an acetaldehyde-derived DNA adduct generated from the reduction of acetaldehyde with 2Ј-deoxyguanosine-3Ј-monophosphate (1). Humans are exposed to acetaldehyde from the environment and through the formation of acetaldehyde by the oxidation of ethanol (2). N 2 -Ethyl-Gua has been detected in the DNA of both alcoholic and nonalcohol drinkers (2, 3). Ethanol is classified as a human carcinogen, and acetaldehyde is known to contribute to the formation of malignant tumors (4). The formation of N 2 -ethylGua during the reduction of acetaldehyde could cause ethanolrelated cancers (5).The ethyl moiety of N 2 -ethyl-Gua is predicted to project into the minor groove of duplex DNA. The N 2 -ethyl-Gua adduct is a strong block to DNA replication by replicative DNA polymerases in vitro and in cells (6, 7). Structures of bacteriophage DNA polymerase (pol) RB69, a homolog of human DNA pol ␣, indicate a possible mechanism of N 2 -ethyl-Gua blocked DNA replication. The structures reveal a DNA-binding motif that contacts the DNA minor groove and functions as an important safeguard to replication fidelity (8). The blocking of replicative DNA pols by N 2 -ethyl-Gua could arise when the ethyl group, protruding into the minor groove, disrupts protein:DNA contacts involved in the proposed "checking mechanism" (8). N 2 -Ethyl-Gua also has a high mis-coding potential during DNA replication with the Klenow fragment of Escherichia coli DNA po...
The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N 2 -ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3′ termini opposite N 2 -ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N 2 -ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn 2+ compared to Mg 2+ . In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N 2 -ethyl-Gua bypass is Mg 2+ -dependent. Strikingly, Mn 2+ activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N 2 -ethyl-Gua and Gua compared to that detected upon Mg 2+ activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high K m values for dCTP insertion opposite N 2 -ethyl-Gua and Gua with Mn 2+ activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn 2+ compared to Mg 2+ indicating that DNA pol κ preferentially utilizes Mg 2+ activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N 2 -ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn 2+ suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis.
A number of putative purine nucleoside and nucleobase adducts of the diazonium ion derived from 3-hydroxy-N-nitrosomorpholine have been synthesized as dimethylacetals. These are converted, in most cases nearly quantitatively, to the aldehydes, or in two cases to their derivatives, on treatment with mild acid to yield standards for a quantitative investigation of alkylation of purine nucleosides and DNA by the above metabolite of the powerful carcinogen N-nitrosomorpholine. The stability of the resulting nucleobase ethoxyacetaldehyde (EA) adducts has been characterized under a number of conditions with respect to their propensity to decompose. The stabilities, compared to that of the previously characterized adduct of the model benzimidazole, are generally unexceptional. Deposition of adducts on purine nucleosides and DNA were quantified in reactions in which 3-hydroperoxy-N-nitrosomorpholine was reduced to the hydroxy metabolite by a water-soluble phosphine at 21 +/- 2 degrees C. The adduct profile is highly similar to that observed from simpler alpha-hydroxy metabolites of acyclic dialkylnitrosamines, with the three most abundant ethoxyacetaldehyde (EA) adducts in reactions of duplex DNA being N7-EA-Gua approximately O(6)-EA-Gua > N3-EA-Ade. The initial rate kinetics of formation of hydroxyethyl (HE) lesions from the initially formed EA lesions have been determined in the case of the major products in the cases of both the nucleoside and DNA adducts. The rates of formation of HE adducts are accelerated in DNA, relative to the nucleosides in the cases of the N7-EA-Ade, N7-EA-Gua, and O(6)-EA-Gua adducts by factors of 7, 14, and 54, respectively. The initial rates of depurination of the N3-EA-Ade, N7-EA-Gua, and N7-EA-Gua adducts have also been quantified, and they are unexceptional in comparison with what has been previously reported for simple alkyl adducts. The adduct profiles reported here stand in significant contrast to what has been reported previously for structurally closely related alpha-substituted cyclic nitrosamines. In part or whole, this may be due to methodological differences in the conduct of the present and previous reports.
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