RESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development.
Assuntos
Cistos , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Camundongos , Animais , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Proliferação de Células , Doenças Renais Policísticas/metabolismo , Apoptose , Estresse Oxidativo , Cistos/metabolismo , DNA/metabolismo , Rim/metabolismo , Canais de Cátion TRPP/genéticaRESUMO
The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissues. Replication of a site-specific 5ClC in a viral DNA genome results in C â T mutations, which is consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune-cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, nonmutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool by exposing cells to the nucleoside 5-chloro-2'-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. High-resolution sequencing of DNA from MEFs derived from the gptΔ C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG â TA transitions in 5'-GC(Y)-3' contexts (Y = a pyrimidine) were dominant, while TA â CG transitions appeared at a much lower frequency. The high-resolution mutational spectrum of 5CldC revealed a notable similarity to the Catalogue of Somatic Mutations in Cancer mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively. SBS84 is associated with the expression of activation-induced cytidine deaminase (AID), a cytosine deaminase associated with inflammation, as well as immunoglobulin gene diversification during antibody maturation. The similarity between the spectra of AID activation and 5CldC could be coincidental; however, the administration of 5CldC did induce some AID expression in MEFs, which have no inherent expression of its gene. In summary, this work shows that 5CldC induces a distinct pattern of mutations in cells. Moreover, that pattern resembles human mutational signatures induced by inflammatory processes, such as those triggered in certain malignancies.
Assuntos
Desoxicitidina/análogos & derivados , Fibroblastos , Neoplasias , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fibroblastos/metabolismo , Mutação , Neoplasias/genética , DNA/metabolismo , Mutagênicos , NucleotídeosRESUMO
This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly AâT transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.
Assuntos
Escherichia coli , Timina , Pareamento de Bases , DNA/genética , Reparo do DNA , Escherichia coli/genéticaRESUMO
DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct lesions that can be both toxic and mutagenic. Foremost among the mutagenic lesions is O6-methylguanine (m6G), which base pairs with thymine during replication to cause GC â AT mutations. The gpt delta C57BL/6J mouse strain of Nohmi et al. (Mol. Mutagen 1996, 28, 465-70) reliably produces mutational spectra of many DNA damaging agents. In this work, mouse embryo fibroblasts (MEFs) were made from gpt delta C57BL/6J mice and evaluated as a screening tool to determine the qualitative and quantitative features of mutagenesis by N-methyl-N-nitrosourea (MNU), a direct-acting DNA alkylator that serves as a model for environmental N-nitrosamines, such as N-nitrosodimethylamine and therapeutic agents such as Temozolomide. The DNA repair protein MGMT (O6-methylguanine DNA methyltransferase) protects against environmental mutagenesis by DNA methylating agents and, by removing m6G, limits the therapeutic potential of Temozolomide in cancer therapy. The gpt delta MEFs were treated with MNU to establish dose-dependent toxicity. In parallel, MNU mutagenicity was determined in the presence and absence of the MGMT inhibitor AA-CW236 (4-(2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole). With and without the inhibitor, the principal mutagenic event of MNU was GC â AT, but more mutations were observed when the inhibitor was present. Evidence that the mutagenic lesion was m6G was based on mass spectral data collected using O6-methyl-d3-guanine as an internal standard; m6G levels were higher in AA-CW236 treated MEFs by an amount proportional to the higher mutation frequency seen in the same cells. This work establishes gpt delta MEFs as a versatile tool for probing mutagenesis by environmental and therapeutic agents and as a cell culture model in which chemical genetics can be used to determine the impact of DNA repair on biological responses to DNA damaging agents.
Assuntos
Alquilantes/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Metilnitrosoureia/farmacologia , Mutagênese/efeitos dos fármacos , Proteínas Supressoras de Tumor/antagonistas & inibidores , Alquilantes/química , Animais , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Inibidores Enzimáticos/química , Fibroblastos/metabolismo , Metilnitrosoureia/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Aflatoxin B1 (AFB1) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB1-DNA adducts in hepatocytes seeds a population of mutations, mainly G:CâT:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:CâT:A mutations characteristic of AFB1 exposure. Importantly, 25% of all mutations were GâT in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB1, and, as such, is an early detection metric for AFB1-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.
Assuntos
Aflatoxina B1/genética , Biomarcadores/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Adutos de DNA/genética , Neoplasias Hepáticas/genética , Mutação , Aflatoxina B1/toxicidade , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/patologia , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Adutos de DNA/toxicidade , Feminino , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
Antiviral drugs designed to accelerate viral mutation rates can drive a viral population to extinction in a process called lethal mutagenesis. One such molecule is 5,6-dihydro-5-aza-2'-deoxycytidine (KP1212), a selective mutagen that induces A-to-G and G-to-A mutations in the genome of replicating HIV. The mutagenic property of KP1212 was hypothesized to originate from its amino-imino tautomerism, which would explain its ability to base pair with either G or A. To test the multiple tautomer hypothesis, we used 2D IR spectroscopy, which offers subpicosecond time resolution and structural sensitivity to distinguish among rapidly interconverting tautomers. We identified several KP1212 tautomers and found that >60% of neutral KP1212 is present in the enol-imino form. The abundant proportion of this traditionally rare tautomer offers a compelling structure-based mechanism for pairing with adenine. Additionally, the pKa of KP1212 was measured to be 7.0, meaning a substantial population of KP1212 is protonated at physiological pH. Furthermore, the mutagenicity of KP1212 was found to increase dramatically at pH <7, suggesting a significant biological role for the protonated KP1212 molecules. Overall, our data reveal that the bimodal mutagenic properties of KP1212 result from its unique shape shifting ability that utilizes both tautomerization and protonation.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/toxicidade , Desoxicitidina/análogos & derivados , Prótons , Sequência de Bases , DNA/química , DNA/genética , Desoxicitidina/química , Desoxicitidina/toxicidade , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Testes de Mutagenicidade , Mutação/genética , Teoria Quântica , Espectroscopia de Infravermelho com Transformada de Fourier , Estereoisomerismo , Temperatura , Água/químicaRESUMO
During chronic inflammation, neutrophil-secreted hypochlorous acid can damage nearby cells inducing the genomic accumulation of 5-chlorocytosine (5ClC), a known inflammation biomarker. Although 5ClC has been shown to promote epigenetic changes, it has been unknown heretofore if 5ClC directly perpetrates a mutagenic outcome within the cell. The present work shows that 5ClC is intrinsically mutagenic, both in vitro and, at a level of a single molecule per cell, in vivo. Using biochemical and genetic approaches, we have quantified the mutagenic and toxic properties of 5ClC, showing that this lesion caused CâT transitions at frequencies ranging from 3-9% depending on the polymerase traversing the lesion. X-ray crystallographic studies provided a molecular basis for the mutagenicity of 5ClC; a snapshot of human polymerase ß replicating across a primed 5ClC-containing template uncovered 5ClC engaged in a nascent base pair with an incoming dATP analog. Accommodation of the chlorine substituent in the template major groove enabled a unique interaction between 5ClC and the incoming dATP, which would facilitate mutagenic lesion bypass. The type of mutation induced by 5ClC, the CâT transition, has been previously shown to occur in substantial amounts both in tissues under inflammatory stress and in the genomes of many inflammation-associated cancers. In fact, many sequence-specific mutational signatures uncovered in sequenced cancer genomes feature CâT mutations. Therefore, the mutagenic ability of 5ClC documented in the present study may constitute a direct functional link between chronic inflammation and the genetic changes that enable and promote malignant transformation.
Assuntos
Citosina/análogos & derivados , Mutagênese , Mutagênicos , Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese , Cromatografia Líquida de Alta Pressão , Citosina/química , Análise Mutacional de DNA , Humanos , Ácido Hipocloroso/química , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Modelos Moleculares , Mutação , Oligonucleotídeos/química , Oligonucleotídeos/genética , Peroxidase/metabolismo , Análise de Sequência de DNARESUMO
Heterocyclic nucleic acid bases and their analogs can adopt multiple tautomeric forms due to the presence of multiple solvent-exchangeable protons. In DNA, spontaneous formation of minor tautomers has been speculated to contribute to mutagenic mispairings during DNA replication, whereas in RNA, minor tautomeric forms have been proposed to enhance the structural and functional diversity of RNA enzymes and aptamers. This review summarizes the role of tautomerism in RNA biochemistry, specifically focusing on the role of tautomerism in catalysis of small self-cleaving ribozymes and recognition of ligand analogs by riboswitches. Considering that the presence of multiple tautomers of nucleic acid bases is a rare occurrence, and that tautomers typically interconvert on a fast time scale, methods for studying rapid tautomerism in the context of nucleic acids under biologically relevant aqueous conditions are also discussed.
Assuntos
RNA Catalítico/química , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , Biocatálise , Isomerismo , Modelos Moleculares , Oxirredução , RNA/química , RiboswitchRESUMO
Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (d- and l-2HG) have been found at abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several α-ketoglutarate (αKG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC50 values of d-2HG on the enzymes, and the results have not been correlated to physiologically relevant concentrations of 2HG and αKG in cancer cells. In this work, we performed detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3, and the bacterial AlkB in the presence of d- and l-2HG in both double- and single-stranded DNA contexts. We determined the kinetic parameters of inhibition, including kcat, KM, and Ki. We also correlated the relative concentrations of 2HG and αKG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both d- and l-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 at pathologically relevant concentrations (73-88% for d-2HG and 31-58% for l-2HG inhibition). This work provides a new perspective that the elevation of the d- or l-2HG concentration in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair performed by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.
Assuntos
Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Glutaratos/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/antagonistas & inibidores , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Reparo do DNA , Ensaios Enzimáticos , Glutaratos/análise , Glutaratos/química , Humanos , Concentração Inibidora 50 , Ácidos Cetoglutáricos/análise , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , EstereoisomerismoRESUMO
Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N(2),3-ethenoguanine (N(2),3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N(2),3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N(2),3-εG and its isomer 1,N(2)-ethenoguanine (1,N(2)-εG) were evaluated in various repair and replication backgrounds. We found that N(2),3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N(2)-εG induces various substitutions and frameshifts. We also found that N(2),3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N(2),3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.
Assuntos
Dano ao DNA , Guanina/análogos & derivados , Mutação , Adutos de DNA/química , DNA Polimerase beta/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Dioxigenases/metabolismo , Guanina/química , Sequenciamento de Nucleotídeos em Larga Escala , Mutagênese , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.
Assuntos
Fármacos Anti-HIV/farmacologia , Azacitidina/análogos & derivados , Desoxicitidina/análogos & derivados , HIV/efeitos dos fármacos , HIV/genética , Mutagênicos/farmacologia , Fármacos Anti-HIV/química , Azacitidina/química , Azacitidina/farmacologia , Bacteriófago M13/efeitos dos fármacos , Bacteriófago M13/genética , Bacteriófago M13/fisiologia , Pareamento de Bases , Desoxicitidina/química , Desoxicitidina/farmacologia , Genoma Viral/efeitos dos fármacos , HIV/fisiologia , Humanos , Isomerismo , Espectroscopia de Ressonância Magnética , Modelos Químicos , Mutagênicos/química , Espectrofotometria Infravermelho , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins.
Assuntos
Reparo do DNA , Dioxigenases/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/metabolismo , Homólogo AlkB 4 da Lisina Desmetilase , Alquilação , Dano ao DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Dioxigenases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Oxigenases de Função Mista/genética , Modelos Moleculares , Família Multigênica , Oxirredução , Especificidade por SubstratoRESUMO
The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.
Assuntos
Acroleína/química , Adutos de DNA/metabolismo , Desoxiguanosina/química , Proteínas de Escherichia coli/metabolismo , Malondialdeído/química , Oxigenases de Função Mista/metabolismo , Boroidretos/química , Cromatografia Líquida de Alta Pressão , DNA/química , DNA/metabolismo , Adutos de DNA/química , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Oligonucleotídeos/análise , Oligonucleotídeos/síntese química , Espectrometria de Massas em TandemRESUMO
The AlkB enzyme is an Fe(II)- and α-ketoglutarate-dependent dioxygenase that repairs DNA alkyl lesions by a direct reversal of damage mechanism as part of the adaptive response in E. coli. The reported substrate scope of AlkB includes simple DNA alkyl adducts, such as 1-methyladenine, 3-methylcytosine, 3-ethylcytosine, 1-methylguanine, 3-methylthymine, and N(6)-methyladenine, as well as more complex DNA adducts, such as 1,N(6)-ethenoadenine, 3,N(4)-ethenocytosine, and 1,N(6)-ethanoadenine. Previous studies have revealed, in a piecemeal way, that AlkB has an impressive repertoire of substrates. The present study makes two additions to this list, showing that alkyl adducts on the N(2) position of guanine and N(4) position of cytosine are also substrates for AlkB. Using high resolution ESI-TOF mass spectrometry, we show that AlkB has the biochemical capability to repair in vitro N(2)-methylguanine, N(2)-ethylguanine, N(2)-furan-2-yl-methylguanine, N(2)-tetrahydrofuran-2-yl-methylguanine, and N(4)-methylcytosine in ssDNA but not in dsDNA. When viewed together with previous work, the experimental data herein demonstrate that AlkB is able to repair all simple N-alkyl adducts occurring at the Watson-Crick base pairing interface of the four DNA bases, confirming AlkB as a versatile gatekeeper of genomic integrity under alkylation stress.
Assuntos
Citosina/análogos & derivados , DNA de Cadeia Simples/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanina/análogos & derivados , Oxigenases de Função Mista/metabolismo , Alquilação , Pareamento de Bases , Cromatografia Líquida de Alta Pressão , Citosina/metabolismo , Adutos de DNA/química , Adutos de DNA/metabolismo , DNA de Cadeia Simples/química , Escherichia coli/enzimologia , Guanina/metabolismo , Oligonucleotídeos/análise , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O 6-methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of â¼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GCâAT mutations in 5'-Pu-G-3' contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.
RESUMO
The antitumor agent 11ß (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11ß against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11ß; flow cytometry studies showed that 11ß exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11ß inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11ß blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11ß enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11ß, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11ß, which supplements conventional DNA adduct formation to promote cancer cell death.
Assuntos
Mostarda de Anilina/farmacologia , Antineoplásicos Alquilantes/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias Hepáticas/metabolismo , Acetilcisteína/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Adutos de DNA/metabolismo , Feminino , Sequestradores de Radicais Livres/farmacologia , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Nus , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vitamina E/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
DNA (2'-deoxyribonucleic acid) and RNA (ribonucleic acid) play diverse functional roles in biology and disease. Despite being comprised primarily of only four cognate nucleobases, nucleic acids can adopt complex three-dimensional structures, and RNA in particular, can catalyze biochemical reactions to regulate a wide variety of biological processes. Such chemical versatility is due in part to the phenomenon of nucleobase tautomerism, whereby the bases can adopt multiple, yet distinct isomeric forms, known as tautomers. For nucleobases, tautomers refer to structural isomers that differ from one another by the position of protons. By altering the position of protons on nucleobases, many of which play critical roles for hydrogen bonding and base pairing interactions, tautomerism has profound effects on the biochemical processes involving nucleic acids. For example, the transient formation of minor tautomers during replication could generate spontaneous mutations. These mutations could arise from the stabilization of mismatches, in the active site of polymerases, in conformations involving minor tautomers that are indistinguishable from canonical base pairs. In this review, we discuss the evidence for tautomerism in DNA, and its consequences to the fidelity of DNA replication. Also reviewed are RNA systems, such as the riboswitches and self-cleaving ribozymes, in which tautomerism plays a functional role in ligand recognition and catalysis, respectively. We also discuss tautomeric nucleoside analogs that are efficacious as antiviral drug candidates such as molnupiravir for coronaviruses and KP1212 for HIV. The antiviral efficacy of these analogs is due, in part, to their ability to exist in multiple tautomeric forms and induce mutations in the replicating viral genomes. From a technical standpoint, minor tautomers of nucleobases are challenging to identify directly because they are rare and interconvert on a fast, millisecond to nanosecond, time scale. Nevertheless, many approaches including biochemical, structural, computational and spectroscopic methods have been developed to study tautomeric dynamics in RNA and DNA systems, and in antiviral nucleoside analogs. An overview of these methods and their applications is included here.
RESUMO
In a multicellular organism, somatic mutations represent a permanent record of the past chemical and biochemical perturbations experienced by a cell in its local microenvironment. Akin to a perpetual recording device, with every replication, genomic DNA accumulates mutations in patterns that reflect: i) the sequence context-dependent formation of DNA damage, due to environmental or endogenous reactive species, including spontaneous processes; ii) the activity of DNA repair pathways, which, depending on the type of lesion, can erase, ignore or exacerbate the mutagenic consequences of that DNA damage; and iii) the choice of replication machinery that synthesizes the nascent genomic copy. These three factors result in a richly contoured sequence context-dependent mutational spectrum that, from appearances, is distinct for most individual forms of DNA damage. Such a mutagenic legacy, if appropriately decoded, can reveal the local history of genome-altering events such as chemical or pathogen exposures, metabolic stress, and inflammation, which in turn can provide an indication of the underlying causes and mechanisms of genetic disease. Modern tools have positioned us to develop a deep mechanistic understanding of the cellular factors and pathways that modulate a mutational process and, in turn, provide opportunities for better diagnostic and prognostic biomarkers, better exposure risk assessment and even actionable therapeutic targets. The goal of this Perspective is to present a bottom-up, lesion-centric framework of mutagenesis that integrates the contributions of lesion replication, lesion repair and lesion formation to explain the complex mutational spectra that emerge in the genome following exposure to mutagens. The mutational spectra of the well-studied hepatocarcinogen aflatoxin B1 are showcased here as specific examples, but the implications are meant to be generalizable.
Assuntos
Aflatoxina B1/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Replicação do DNA , Mutagênese , Aflatoxina B1/farmacologia , Aflatoxina B1/toxicidade , Animais , Bactérias/genética , Bactérias/metabolismo , Carcinógenos Ambientais/toxicidade , DNA/efeitos dos fármacos , Humanos , MutaçãoRESUMO
Paraquat (1,1'-dimethyl, 4,4'-bipyridinium dichloride; PQ), a widely used herbicide, is toxic to mammals through ingestion, inhalation and skin contact. Epidemiological data suggest that PQ is also mutagenic and carcinogenic, especially in high doses. The toxic and mutagenic properties of PQ are attributed to the ability of the molecule to redox-cycle, which generates reactive oxygen species (ROS) and subsequent oxidative stress. ROS also cause oxidative DNA damage such as 8-oxoguanine (8OG), a mutagenic base that, when replicated, causes G to T transversion mutations. The present study employed the CHO-derived cell line AS52 to quantify the mutagenic properties of low doses of PQ. By containing a functional, chromosomally-integrated copy of the bacterial gpt gene, AS52 cells a facile system for evaluating the mutagenic properties of genotoxicants. To bolster the sensitivity of this system for detecting mutagenesis of weak mutagens like PQ, and to provide a tool for mechanistic evaluation of the mutagenic process, we constructed a new AS52-derived cell line defective for 8OG DNA repair. Specifically, we employed CRISPR-Cas9 technology to knock out 8-oxoguanine DNA glycosylase (OGG1) and MUTYH glycosylase, two key enzymes involved in the base excision repair of 8OG. The double knock-out (DKO) AS52 cells were found to be more sensitive to PQ toxicity than the parental (WT) AS52 cell line. They experienced higher levels of ROS, which translated into more DNA double-strand breaks, which explained the PQ toxicity. The increased ROS levels also led to more 8OG genomic accumulation, and a higher level of mutations in the DKO cells, suggesting that PQ mutagenesis is mediated primarily by 8OG genomic accumulation. Consistent with this view, antioxidant co-treatment lowered induced cellular ROS and PQ-induced mutagenesis. Taken together, our data demonstrate the strong protective role of OGG1 and MUTYH against PQ-induced mutagenesis. Moreover, our experiments establish the engineered OGG1-/-MUTYH-/- AS52 cell line and associated methods as a versatile cellular system for studying in quantitative terms the mutagenesis of other agents, environmental or endogenous, that induce oxidative stress.