DNA Strand Cleavage Research Articles

Toward discovering new anti-cancer agents targeting topoisomerase IIα: a facile screening strategy adaptable to high throughput platform.

Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α). The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α–mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.

Synthesis and characterization of new transition metal {Cu(II), Ni(II) and Co(II)} l-phenylalanine–DACH conjugate complexes: In vitro DNA binding, cleavage and molecular docking studies

The novel metal-based molecular entities {Cu(II), Ni(II) and Co(II)} 1–3, respectively were synthesized and characterized by elemental analysis and spectroscopic methods (IR, 1H and 13C NMR, EPR, UV–vis, ESI–MS and XRPD). The interaction studies of 1–3 with CT DNA have been investigated by UV–vis titrations, fluorescence and circular dichroic studies which revealed the electrostatic mode of binding and on the basis of intrinsic binding constant Kb (5.30×104, 1; 3.41×104, 2; and 2.74×104, 3; M−1), the extent of DNA binding was ascertained, following the order 1>2>3. Specifically, 1 exhibited greater binding propensity with CT DNA, therefore the cleavage activity of 1 with pBR322 DNA was evaluated by agarose gel electrophoresis assay. Complex 1 presented an impressive nuclease activity generating single- and double-strand breaks. Further mechanistic investigation revealed the efficiency of 1 to cleave DNA strands by an oxidative pathway involving the generation of ROS and preferential selectivity towards the DNA minor groove. Moreover, complex 1 exhibited significant inhibitory effects on the catalytic activity of topoisomerase I at a very low concentration ∼20μM. Additionally, computer-aided molecular docking techniques were carried out to correlate and rationalize the observed binding affinities with docking studies towards the molecular target DNA and Topo-I.

Considerations on the Mechanism of Action of Artemisinin Antimalarials: Part 1 - The 'Carbon Radical' and 'Heme' Hypotheses

Considerations on the Mechanism of Action of Artemisinin Antimalarials: Part 1 - The 'Carbon Radical' and 'Heme' Hypotheses

21pm3-PM024 DNAナノマシンによる歩行動作の温度特性の検証

In the recent years, construction of walking nanomachines that utilize the self-assembly of DNA molecules, coupled with the enzymatic reaction to cleave DNA strands in a sequence-specific manner, has been reported. However, the conventional walking DNA nanomachines have some limitations on reaction conditions due to their reaction components. For achieving the versatile nanomechanical systems that are applicable to control chemical processes, elementary DNA nanomachines are expected to work under various reaction conditions. In this study, we propose a novel type of walking DNA nanomachine, and experimentally investigated the thermal property of its elementary reaction to discuss the walking performance of the proposed DNA nanomachine.

Isotopic Labeling Experiments That Elucidate the Mechanism of DNA Strand Cleavage by the Hypoxia-Selective Antitumor Agent 1,2,4-Benzotriazine 1,4-Di-N-oxide

The 1,2,4-benzotriazine 1,4-dioxides are an important class of potential anticancer drugs that selectively kill the low-oxygen (hypoxic) cells found in solid tumors. These compounds undergo intracellular one-electron enzymatic reduction to yield an oxygen-sensitive drug radical intermediate that partitions forward, under hypoxic conditions, to generate a highly reactive secondary radical that causes cell killing DNA damage. Here, we characterized bioreductively activated, hypoxia-selective DNA-strand cleavage by 1,2,4-benzotriazine 1,4-dioxide. We found that one-electron enzymatic activation of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions in the presence of the deuterium atom donor methanol-d4 produced nondeuterated mono-N-oxide metabolites. This and the results of other isotopic labeling studies provided evidence against the generation of atom-abstracting drug radical intermediates and are consistent with a DNA-damage mechanism involving the release of hydroxyl radical from enzymatically activated 1,2,4-benzotriazine 1,4-dioxides.

Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

BackgroundDNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.MethodsThe three resistant mutants have been investigated in S. cerevisiae model system following their viability in presence of increasing amounts of camptothecin. A systematical analysis of the different catalytic steps has been made for one of these mutants (Glu710Gly) and has been correlated with its structural-dynamical properties studied by classical molecular dynamics simulation.ResultsThe three mutants display a different degree of camptothecin resistance in a yeast cell viability assay. Characterization of the different steps of the catalytic cycle of the Glu710Gly mutant indicated that its resistance is related to a high religation rate that is hardly affected by the presence of the drug. Analysis of the dynamic properties through simulation indicate that the mutant displays a much lower degree of correlation in the motion between the different protein domains and that the linker almost completely loses its correlation with the C-terminal domain, containing the active site tyrosine.ConclusionsThese results indicate that a fully functional linker is required to confer camptothecin sensitivity to topoisomerase I since the destabilization of its structural-dynamical properties is correlated to an increase of religation rate and drug resistance.

TOP2A overexpression as a poor prognostic factor in patients with nasopharyngeal carcinoma

Despite the advances in diagnostic imaging and treatment modalities, the risk stratification and final outcomes in patients with nasopharyngeal carcinomas (NPC) still remain suboptimal. Through data mining from published transcriptomic database, topoisomerase IIα (TOP2A) was first identified as a differentially upregulated gene in NPC tissues, which implicates cell division via selective cleavage, rearrangement, and re-ligation of DNA strands. Given the roles of TOP2A in prognostication and in the frontline therapeutic regimen of common carcinomas, such as breast cancer, we explored TOP2A immunoexpression status and its associations with clinicopathological variables and survival in a well-defined cohort of NPC patients. TOP2A immunohistochemistry was retrospectively performed and analyzed using H-score method for biopsy specimens from 124 NPC patients who received standard treatment without distant metastasis at initial diagnosis. Those cases with H-score larger than the median value were construed as featuring TOP2A overexpression. The findings were correlated with the clinicopathological variables, disease-specific survival (DSS) and distant metastasis-free survival (DMFS). TOP2A overexpression was significantly associated with American Joint of Cancer Committee (AJCC) stages III-IV (p = 0.019) and univariately predictive of adverse outcomes for DSS (p = 0.0078) and DMFS (p = 0.0003). In the multivariate comparison, TOP2A overexpression remained prognostically independent to portend worse DSS (p = 0.047, hazard ratio = 1.732) and DMFS (p = 0.003, hazard ratio = 2.569), together with advanced AJCC stages III-IV. TOP2A expression is upregulated in a subset of NPCs and its increased immunoexpression significantly correlated with advanced stages and tumor aggressiveness, justifying the potentiality of TOP2A as a prognostic biomarker and a novel therapeutic target of NPC.

The role of DNA bending in type IIA topoisomerase function

Type IIA topoisomerases control DNA supercoiling and separate newly replicated chromosomes using a complex DNA strand cleavage and passage mechanism. Structural and biochemical studies have shown that these enzymes sharply bend DNA by as much as 150°; an invariant isoleucine, which has been seen structurally to intercalate between two base pairs outside of the DNA cleavage site, has been suggested to promote deformation. To test this assumption, we examined the role of isoleucine on DNA binding, bending and catalytic activity for a bacterial type IIA topoisomerase, Escherichia coli topoisomerase IV (topo IV), using a combination of site-directed mutagenesis and biochemical assays. Our data show that alteration of the isoleucine (Ile172) did not affect the basal ATPase activity of topo IV or its affinity for DNA. However, the amino acid was important for DNA bending, DNA cleavage and supercoil relaxation. Moreover, an ability to bend DNA correlated with efficacy with which nucleic acid substrates stimulate ATP hydrolysis. These data show that DNA binding and bending by topo IV can be uncoupled, and indicate that the stabilization of a highly curved DNA geometry is critical to the type IIA topoisomerase catalytic cycle.

Photoinduced DNA cleavage by atomic oxygen precursors in aqueous solutions

Reactive oxygen species are known to induce DNA strand cleavage and have been explored as treatments for cancer. The development of aqueous-soluble dibenzothiophene-S-oxide (DBTO) derivatives has made it possible to investigate the mechanism of DNA cleavage by these photoactivatable precursors of atomic oxygen. In addition to the release of atomic oxygen, DBTO can also undergo other processes such as α-cleavage. An objective of this work was to establish whether the extent of strand scission could be attributed to a direct reaction between atomic oxygen and DNA. To accomplish this aim, the extent of strand cleavage upon irradiation of three different DBTO derivatives was measured by the conversion of circular pUC19 plasmid (Form I) to nicked (Form II) as monitored by gel electrophoresis. The interaction of the sulfoxides with DNA was systematically studied by optical melt and fluorescence anisotropy experiments. Thiols are susceptible to rapid oxidation by atomic oxygen, and thus, glutathione was used as a ROS scavenger to determine if DNA cleavage was induced by the release of atomic oxygen. The results from these experiments indicated atomic oxygen was at least partially responsible for the observed strand scission.

Crystal structure of an intermediate of rotating dimers within the synaptic tetramer of the G-segment invertase.

The serine family of site-specific DNA recombination enzymes accomplishes strand cleavage, exchange and religation using a synaptic protein tetramer. A double-strand break intermediate in which each protein subunit is covalently linked to the target DNA substrate ensures that the recombination event will not damage the DNA. The previous structure of a tetrameric synaptic complex of γδ resolvase linked to two cleaved DNA strands had suggested a rotational mechanism of recombination in which one dimer rotates 180° about the flat exchange interface for strand exchange. Here, we report the crystal structure of a synaptic tetramer of an unliganded activated mutant (M114V) of the G-segment invertase (Gin) in which one dimer half is rotated by 26° or 154° relative to the other dimer when compared with the dimers in the synaptic complex of γδ resolvase. Modeling shows that this rotational orientation of Gin is not compatible with its being able to bind uncleaved DNA, implying that this structure represents an intermediate in the process of strand exchange. Thus, our structure provides direct evidence for the proposed rotational mechanism of site-specific recombination.

The emerging diversity of transpososome architectures

DNA transposases are enzymes that catalyze the movement of discrete pieces of DNA from one location in the genome to another. Transposition occurs through a series of controlled DNA strand cleavage and subsequent integration reactions that are carried out by nucleoprotein complexes known as transpososomes. Transpososomes are dynamic assemblies which must undergo conformational changes that control DNA breaks and ensure that, once started, the transposition reaction goes to completion. They provide a precise architecture within which the chemical reactions involved in transposon movement occur, but adopt different conformational states as transposition progresses. Their components also vary as they must, at some stage, include target DNA and sometimes even host-encoded proteins. A very limited number of transpososome states have been crystallographically captured, and here we provide an overview of the various structures determined to date. These structures include examples of DNA transposases that catalyze transposition by a cut-and-paste mechanism using an RNaseH-like nuclease catalytic domain, those that transpose using only single-stranded DNA substrates and targets, and the retroviral integrases that carry out an integration reaction very similar to DNA transposition. Given that there are a number of common functional requirements for transposition, it is remarkable how these are satisfied by complex assemblies that are so architecturally different.

Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution.

Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the ‘nick-counter-nick’ mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.

Synthesis and characterization of a small analogue of the anticancer natural product leinamycin

Leinamycin (1) is a Streptomyces-derived natural product that displays nanomolar IC50 values against human cancer cell lines. In the work described here, we report the synthesis and characterization of a small leinamycin analogue 19 that closely resembles the ‘upper-right quadrant’ of the natural product, consisting of an alicyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an alkene by a two-carbon linker. The results indicate that this small analogue contains the core set of functional groups required to enable thiol-triggered generation of both redox active polysulfides and an episulfonium ion intermediate via the complex reaction cascade first seen in the natural product leinamycin. The small leinamycin analogue 19 caused thiol-triggered oxidative DNA strand cleavage in a manner similar to the natural product, but did not alkyate duplex DNA effectively. This highlights the central role of the 18-membered macrocycle of leinamycin in driving efficient DNA alkylation by the natural product.

Ultrasensitive electrogenerated chemiluminescent DNA-based biosensing switch for the determination of bleomycin

An ultrasensitive electrogenerated chemiluminescent (ECL) DNA-based biosensing switch for the determination of bleomycin (BLM) was developed based on Fe(II) ·BLM-mediated hairpin DNA strand cleavage and a structure-switching ECL-dequenching mechanism. A thiolated ss-DNA was used as a substrate for BLMs: one terminus was tethered onto an electrode surface, and the other terminus was labelled with the ECL quencher ferrocene to form a hairpin structure. This thiolated ss-DNA self-assembled on to the tris(2,2′-bipyridine)ruthenium-gold nanoparticle composite modified gold electrode. In the presence of Fe(II) ·BLM, the ECL DNA biosensing switch undergoes an irreversible cleavage event that can trigger a significant increase in ECL intensity. The relationship of ECL intensity and the concentration of BLMs was found to be linear in the range of 5fM – 5000fM with a detection limit of 2fM. This work demonstrates that the design of a highly sensitive ECL DNA-based biosensing switch that uses the sequence selectivity of DNA cleavage mediated by the antitumor drug BLM in combination with a chemical quencher, such as ferrocene, to quench ECL signal(s), offers a promising approach for the determination of ultratrace amounts of antitumor drugs.

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K(d) = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15-44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-L-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H(2)O(2) which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H(2)O(2) significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H(2)O(2) significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H(2)O(2). NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H(2)O(2).

Effects of curcumin on bleomycin-induced oxidative stress in malignant testicular germ cell tumors

Bleomycin is commonly used in the treatment of testicular cancer. Bleomycin generates oxygen radicals, induces the oxidative cleavage of DNA strands and induces cancer cell apoptosis. Curcumin (diferuloylmethane) is a potent antioxidant and chief component of the spice turmeric. No study investigating the effects of curcumin on intrinsic and bleomycin-induced oxidative stress in testicular germ cell tumors has been reported in the literature. For this reason, the present study aimed to examine the effects of curcumin on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testicular cancer cells incubated with bleomycin and the results were compared with cells treated with H2O2 which directly produces oxidative stress. The protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide (LPO) levels and total antioxidant capacity in the two testicular cancer cell lines were determined. Results showed that bleomycin and H2O2 significantly increased protein carbonyl, TBARS, 8-isoprostane and LPO levels in the NTera-2 and NCCIT cell lines. Bleomycin and H2O2 significantly decreased the antioxidant capacity and GSH levels in NTera-2 cells. Curcumin significantly decreased LPO, 8-isoprostane and protein carbonyl content, and TBARS levels increased in cells treated with bleomycin and H2O2. Curcumin enhanced GSH levels and the antioxidant capacity of NTera-2 cells. In conclusion, curcumin inhibits bleomycin and H2O2-induced oxidative stress in human testicular cancer cells.

Signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A

In this work, a new signal amplified strategy based on target-induced strand release coupling cleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A (OTA) is reported. OTA aptamer (DNA1) and OTA aptamer complementary (DNA2) were immobilized onto a magnetic bead (MB). In the presence of OTA, DNA2 was dissociated and released from the MB. The released DNA2 then hybridized with DNA3, which was linked at the 5' terminus of the amplification template and can extend along the template in the presence of Phi 29 DNA polymerase. The formed double-stranded DNA was cleaved by nicking endonuclease Nb.BbvCI and produced a short single-stranded DNA. The cleaved DNA strand generated a new site by Phi 29 DNA polymerase and the process of extension and cleavage was cyclical. Thus, a amount of the short single-stranded DNA were produced. Using DNA and ABEI labeled carboxylic silica nanoparticles chemiluminescence (CL) probe, the short single-stranded DNA could be sensitively detected. The CL intensity (ΔI) versus the concentration of OTA was linear in the range from 1.0×10−12 to 5.0×10−8gmL−1. The detection limit was 3.0×10−13gmL−1, and the RSD was 3.4% at 1.0×10−10gmL−1 (n=7). The developed method has been applied to detect OTA in naturally contaminated wheat samples. Due to its simplicity, sensitivity and no need of specific recognition of aptamer for cleavage, this CL bioassay offers a promising approach for the detection of OTA and other biomolecules.

Generation of DNA-Damaging Reactive Oxygen Species via the Autoxidation of Hydrogen Sulfide under Physiologically Relevant Conditions: Chemistry Relevant to Both the Genotoxic and Cell Signaling Properties of H2S

Hydrogen sulfide (H(2)S) has long been known for its toxic properties; however, in recent years, evidence has emerged that this small, gaseous molecule may serve as an endogenous cell-signaling agent. Though perhaps surprising in light of its potential role as an endogenous signaling agent, a number of studies have provided evidence that H(2)S is a DNA-damaging mutagen. In the work reported here, the chemical mechanisms of DNA damage by H(2)S were examined. Using a plasmid-based DNA strand cleavage assay, we found that micromolar concentrations of H(2)S generated single-strand DNA cleavage. Mechanistic studies indicate that this process involved autoxidation of H(2)S to generate superoxide, hydrogen peroxide, and, ultimately, the well-known DNA-damaging agent hydroxyl radical via a trace metal-mediated Fenton-type reaction. Strand cleavage by H(2)S proceeded in the presence of physiological thiol concentrations, and the known byproducts of H(2)S oxidation such as thiosulfate, sulfite, and sulfate do not contribute to the strand cleavage process. However, initially generated oxidation products such as persulfide (S(2)(2-)) likely undergo rapid autoxidation reactions that contribute to the generation of superoxide. The potential relevance of autoxidation processes to the genotoxic and cell signaling properties of H(2)S is discussed.