RESUMO
Alkylating agents damage DNA and proteins and are widely used in cancer chemotherapy. While cellular responses to alkylation-induced DNA damage have been explored, knowledge of how alkylation affects global cellular stress responses is sparse. Here, we examined the effects of the alkylating agent methylmethane sulfonate (MMS) on gene expression in mouse liver, using mice deficient in alkyladenine DNA glycosylase (Aag), the enzyme that initiates the repair of alkylated DNA bases. MMS induced a robust transcriptional response in wild-type liver that included markers of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) known to be controlled by XBP1, a key UPR effector. Importantly, this response is significantly reduced in the Aag knockout. To investigate how AAG affects alkylation-induced UPR, the expression of UPR markers after MMS treatment was interrogated in human glioblastoma cells expressing different AAG levels. Alkylation induced the UPR in cells expressing AAG; conversely, AAG knockdown compromised UPR induction and led to a defect in XBP1 activation. To verify the requirements for the DNA repair activity of AAG in this response, AAG knockdown cells were complemented with wild-type Aag or with an Aag variant producing a glycosylase-deficient AAG protein. As expected, the glycosylase-defective Aag does not fully protect AAG knockdown cells against MMS-induced cytotoxicity. Remarkably, however, alkylation-induced XBP1 activation is fully complemented by the catalytically inactive AAG enzyme. This work establishes that, besides its enzymatic activity, AAG has noncanonical functions in alkylation-induced UPR that contribute to cellular responses to alkylation.
Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , Desdobramento de Proteína , Alquilação , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Estresse do Retículo Endoplasmático , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-ß-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the 'HepaCometChip', enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.
Assuntos
Ensaio Cometa/métodos , Adutos de DNA/análise , Carcinogênese , Linhagem Celular , Quebras de DNA de Cadeia Simples , Reparo do DNA , Humanos , Análise em Microsséries/métodos , Sensibilidade e EspecificidadeRESUMO
Programmed necrosis has emerged as a crucial modulator of cell death in response to several forms of cellular stress. In one form of programmed necrotic cell death, induced by cytotoxic alkylating agents, hyperactivation of poly-ADP-ribose polymerase (PARP) leads to cellular NAD and ATP depletion, mitochondrial dysfunction, reactive oxygen species formation, and ensuing cell death. Here, we show that the protein encoded by the human AlkB homolog 7 (ALKBH7) gene plays a pivotal role in DNA-damaging agent-induced programmed necrosis by triggering the collapse of mitochondrial membrane potential and large-scale loss of mitochondrial function that lead to energy depletion and cellular demise. Depletion of ALKBH7 suppresses necrotic cell death induced by numerous alkylating and oxidizing agents while having no effect on apoptotic cell death. Like wild-type cells, ALKBH7-depleted cells undergo PARP hyperactivation and NAD depletion after severe DNA damage but, unlike wild-type cells, exhibit rapid recovery of intracellular NAD and ATP levels. Consistent with the recovery of cellular bioenergetics, ALKBH7-depleted cells maintain their mitochondrial membrane potential, plasma membrane integrity, and viability. Our results uncover a novel role for a mammalian AlkB homolog in programmed necrosis, presenting a new target for therapeutic intervention in cancer cells that are resistant to apoptotic cell death.
Assuntos
Apoptose , Dano ao DNA , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Trifosfato de Adenosina/metabolismo , Enzimas AlkB , Alquilação , Apoptose/efeitos dos fármacos , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Resistência a Medicamentos , Metabolismo Energético , Ativação Enzimática , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , NAD/metabolismo , Necrose/metabolismo , Necrose/patologia , Proteínas Nucleares/deficiência , Oxirredução , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte ProteicoRESUMO
The integrity of our DNA is challenged with at least 100,000 lesions per cell on a daily basis. Failure to repair DNA damage efficiently can lead to cancer, immunodeficiency, and neurodegenerative disease. Base excision repair (BER) recognizes and repairs minimally helix-distorting DNA base lesions induced by both endogenous and exogenous DNA damaging agents. Levels of BER-initiating DNA glycosylases can vary between individuals, suggesting that quantitating and understanding interindividual differences in DNA repair capacity (DRC) may enable us to predict and prevent disease in a personalized manner. However, population studies of BER capacity have been limited because most methods used to measure BER activity are cumbersome, time consuming and, for the most part, only allow for the analysis of one DNA glycosylase at a time. We have developed a fluorescence-based multiplex flow-cytometric host cell reactivation assay wherein the activity of several enzymes [four BER-initiating DNA glycosylases and the downstream processing apurinic/apyrimidinic endonuclease 1 (APE1)] can be tested simultaneously, at single-cell resolution, in vivo. Taking advantage of the transcriptional properties of several DNA lesions, we have engineered specific fluorescent reporter plasmids for quantitative measurements of 8-oxoguanine DNA glycosylase, alkyl-adenine DNA glycosylase, MutY DNA glycosylase, uracil DNA glycosylase, and APE1 activity. We have used these reporters to measure differences in BER capacity across a panel of cell lines collected from healthy individuals, and to generate mathematical models that predict cellular sensitivity to methylmethane sulfonate, H2O2, and 5-FU from DRC. Moreover, we demonstrate the suitability of these reporters to measure differences in DRC in multiple pathways using primary lymphocytes from two individuals.
Assuntos
Variação Biológica da População/fisiologia , Dano ao DNA/fisiologia , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Linhagem Celular , DNA/genética , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Citometria de Fluxo/métodos , Fluoruracila/toxicidade , Técnicas de Silenciamento de Genes , Voluntários Saudáveis , Humanos , Peróxido de Hidrogênio/toxicidade , Metanossulfonato de Metila/toxicidade , Modelos Biológicos , Mutagênese/efeitos dos fármacos , Mutagênese/fisiologia , Mutagênicos/toxicidade , Medicina de Precisão/métodos , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Análise de Célula Única/métodos , Linfócitos TRESUMO
Following publication of the original article [1], the author reported that incorrect version of Tables 1, 3, 5 and 6 were published.
RESUMO
BACKGROUND: Growing evidence indicates that in utero arsenic exposures in humans may increase the risk of adverse health effects and development of diseases later in life. This study aimed to evaluate potential health risks of in utero arsenic exposure on genetic damage in newborns in relation to maternal arsenic exposure. METHODS: A total of 205 pregnant women residing in arsenic-contaminated areas in Hanam province, Vietnam, were recruited. Prenatal arsenic exposure was determined by arsenic concentration in mother's toenails and urine during pregnancy and in umbilical cord blood collected at delivery. Genetic damage in newborns was assessed by various biomarkers of early genetic effects including oxidative/nitrative DNA damage (8-hydroxydeoxyguanosine, 8-OHdG, and 8-nitroguanine), DNA strand breaks and micronuclei (MN) in cord blood. RESULTS: Maternal arsenic exposure, measured by arsenic levels in toenails and urine, was significantly increased (p < 0.05) in subjects residing in areas with high levels of arsenic contamination in drinking water. Cord blood arsenic level was significantly increased in accordance with maternal arsenic exposure (p < 0.001). Arsenic exposure in utero is associated with genotoxic effects in newborns indicated as increased levels of 8-OHdG, 8-nitroguanine, DNA strand breaks and MN frequency in cord blood with increasing levels of maternal arsenic exposure. Maternal toenail arsenic level was significantly associated with all biomarkers of early genetic effects, while cord blood arsenic levels associated with DNA strand breaks and MN frequency. CONCLUSIONS: In utero arsenic exposure is associated with various types of genetic damage in newborns potentially contributing to the development of diseases, including cancer, later in life.
Assuntos
Arsênio/toxicidade , Dano ao DNA/efeitos dos fármacos , Sangue Fetal/química , Exposição Materna/efeitos adversos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Adulto , Biomarcadores/sangue , Feminino , Humanos , Recém-Nascido , Unhas/química , Gravidez , Vietnã , Adulto JovemRESUMO
Etheno (ε) DNA base adducts are highly mutagenic lesions produced endogenously via reactions with lipid peroxidation (LPO) products. Cancer-promoting conditions, such as inflammation, can induce persistent oxidative stress and increased LPO, resulting in the accumulation of ε-adducts in different tissues. Using a recently described fluorescence multiplexed host cell reactivation assay, we show that a plasmid reporter bearing a site-specific 3,N4-ethenocytosine (εC) causes transcriptional blockage. Notably, this blockage is exacerbated in Cockayne Syndrome and xeroderma pigmentosum patient-derived lymphoblastoid and fibroblast cells. Parallel RNA-Seq expression analysis of the plasmid reporter identifies novel transcriptional mutagenesis properties of εC. Our studies reveal that beyond the known pathways, such as base excision repair, the process of transcription-coupled nucleotide excision repair plays a role in the removal of εC from the genome, and thus in the protection of cells and tissues from collateral damage induced by inflammatory responses.
Assuntos
Citosina/análogos & derivados , Adutos de DNA/metabolismo , Reparo do DNA , Transcrição Gênica , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Síndrome de Cockayne/genética , Citosina/metabolismo , Enzimas Reparadoras do DNA/genética , Humanos , Camundongos , Camundongos Knockout , Mutagênese , RNA Polimerase II/metabolismo , Xeroderma Pigmentoso/genéticaRESUMO
Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine, and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than does the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L-induced mutator phenotype. The hAAG-Y127I/H136L and hAAG-Y127I mutants increased the rate of spontaneous frameshifts by up to 120-fold in S. cerevisiae and also induced high rates of microsatellite instability (MSI) in human cells. hAAG and its mutants bind DNA containing one and two base-pair loops with significant affinity, thus shielding them from mismatch repair; the strength of such binding correlates with their ability to induce the mutator phenotype. This study provides important insights into the mechanism of hAAG-induced genomic instability.
Assuntos
DNA Glicosilases/metabolismo , Mutação da Fase de Leitura , Instabilidade de Microssatélites , Domínio Catalítico , DNA Glicosilases/química , DNA Glicosilases/genética , Reparo de Erro de Pareamento de DNA , Regulação Enzimológica da Expressão Gênica , Humanos , Células K562 , Modelos Moleculares , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The capacity to repair different types of DNA damage varies among individuals, making them more or less susceptible to the detrimental health consequences of damage exposures. Current methods for measuring DNA repair capacity (DRC) are relatively labor intensive, often indirect, and usually limited to a single repair pathway. Here, we describe a fluorescence-based multiplex flow-cytometric host cell reactivation assay (FM-HCR) that measures the ability of human cells to repair plasmid reporters, each bearing a different type of DNA damage or different doses of the same type of DNA damage. FM-HCR simultaneously measures repair capacity in any four of the following pathways: nucleotide excision repair, mismatch repair, base excision repair, nonhomologous end joining, homologous recombination, and methylguanine methyltransferase. We show that FM-HCR can measure interindividual DRC differences in a panel of 24 cell lines derived from genetically diverse, apparently healthy individuals, and we show that FM-HCR may be used to identify inhibitors or enhancers of DRC. We further develop a next-generation sequencing-based HCR assay (HCR-Seq) that detects rare transcriptional mutagenesis events due to lesion bypass by RNA polymerase, providing an added dimension to DRC measurements. FM-HCR and HCR-Seq provide powerful tools for exploring relationships among global DRC, disease susceptibility, and optimal treatment.
Assuntos
Dano ao DNA , Reparo do DNA , Técnicas Genéticas , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Reparo de Erro de Pareamento de DNA , Citometria de Fluxo , Genes Reporter , Guanina/análogos & derivados , Guanina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutagênese , Plasmídeos/genética , Análise de Sequência de RNA , Transcrição Gênica , TransfecçãoRESUMO
Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag(-/-) mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag(-/-) mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag(-/-) liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.
Assuntos
Encéfalo/enzimologia , DNA Glicosilases/metabolismo , Reparo do DNA , Rim/enzimologia , Fígado/enzimologia , Traumatismo por Reperfusão/enzimologia , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Encéfalo/patologia , Infarto Encefálico/enzimologia , Infarto Encefálico/genética , Infarto Encefálico/patologia , Morte Celular , Dano ao DNA , DNA Glicosilases/genética , Indução Enzimática/genética , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Hepatócitos/enzimologia , Hepatócitos/patologia , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Rim/patologia , Fígado/patologia , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologiaRESUMO
Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic ß-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aagâ»/â» mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.
Assuntos
Antineoplásicos Alquilantes , DNA Glicosilases , Neoplasias/tratamento farmacológico , Poli(ADP-Ribose) Polimerases , Alquilação/efeitos dos fármacos , Alquilação/genética , Animais , Antineoplásicos Alquilantes/efeitos adversos , Antineoplásicos Alquilantes/uso terapêutico , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/lesões , Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Timócitos/citologia , Timócitos/efeitos dos fármacosRESUMO
An effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed. Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF. We conclude that cellular responses can navigate different routes according to the extent of damage, relying on both expression and localization changes of specific proteins.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina , Citoplasma/química , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Fúngico , Lipídeos/biossíntese , Metanossulfonato de Metila/toxicidade , Proteínas Nucleares/análise , Biossíntese de Proteínas , Proteólise , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismoRESUMO
Cancer susceptibility varies between people, affected by genotoxic exposures, genetic makeup and physiological state. Yet, how these factors interact among each other to define cancer risk is largely unknown. Here, we uncover the interactive effects of genetical, environmental and physiological factors on genome rearrangements driven by homologous recombination (HR). Using FYDR mice to quantify HR-driven rearrangements in pancreas tissue, we show that DNA methylation damage (induced by methylnitrosourea) and cell proliferation (induced by thyroid hormone) each induce HR and together act synergistically to induce HR-driven rearrangements in vivo. These results imply that developmental or regenerative proliferation as well as mitogenic exposures may sensitize tissues to DNA damaging exposures. We exploited mice genetically deficient in alkyl-adenine DNA glycosylase (Aag) to analyse the relative contributions of unrepaired DNA base lesions versus intermediates formed during base excision repair (BER). Remarkably, results show that, in the pancreas, Aag is a major driver of spontaneous HR, indicating that BER intermediates (including abasic sites and single strand breaks) are more recombinogenic than the spontaneous base lesions removed by Aag. Given that mammals have about a dozen DNA glycosylases, these results point to BER as a major source of pressure on the HR pathway in vivo. Taken together, methylation damage, cell proliferation and Aag interact to define the risk of HR-driven sequence rearrangements in vivo. These data identify important sources of sequence changes in a cancer-relevant organ, and advance the effort to identify populations at high-risk for cancer.
Assuntos
DNA Glicosilases/genética , Reparo do DNA/genética , N-Glicosil Hidrolases/genética , Neoplasias/genética , Animais , Carcinogênese , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Metilação de DNA/genética , Recombinação Homóloga , Humanos , Metilnitrosoureia/toxicidade , Camundongos , Neoplasias/metabolismoAssuntos
Saúde Ambiental/normas , Poluição Ambiental/efeitos adversos , Saúde Global/normas , Adolescente , Adulto , Distribuição por Idade , Idoso , Poluentes Atmosféricos/efeitos adversos , Criança , Pré-Escolar , Doença Crônica/epidemiologia , Doença Crônica/prevenção & controle , Conservação dos Recursos Naturais , Efeitos Psicossociais da Doença , Saúde Ambiental/economia , Saúde Ambiental/legislação & jurisprudência , Poluição Ambiental/prevenção & controle , Feminino , Saúde Global/estatística & dados numéricos , Política de Saúde , Disparidades nos Níveis de Saúde , Humanos , Lactente , Recém-Nascido , Cooperação Internacional , Masculino , Pessoa de Meia-Idade , Mortalidade Prematura , Doenças não Transmissíveis/epidemiologia , Saúde Ocupacional/normas , Pobreza , Características de Residência , Poluentes do Solo/efeitos adversos , Poluentes da Água/efeitos adversos , Organização Mundial da Saúde , Adulto JovemRESUMO
Neurodevelopment is a tightly coordinated process, during which the genome is exposed to spectra of endogenous agents at different stages of differentiation. Emerging evidence indicates that DNA damage is an important feature of developing brain, tightly linked to gene expression and neuronal activity. Some of the most frequent DNA damage includes changes to DNA bases, which are recognized by DNA glycosylases and repaired through base excision repair (BER) pathway. The only mammalian DNA glycosylase able to remove frequent alkylated DNA based is alkyladenine DNA glycosylase (Aag, aka Mpg). We recently demonstrated that, besides its role in DNA repair, AAG affects expression of neurodevelopmental genes in human cells. Aag was further proposed to act as reader of epigenetic marks, including 5-hydroxymethylcytosine (5hmC), in the mouse brain. Despite the potential Aag involvement in the key brain processes, the impact of Aag loss on developing brain remains unknown. Here, by using Aag knockout (Aag-/-) mice, we show that Aag absence leads to reduced DNA break levels, evident in lowered number of γH2AX foci in postnatal day 5 (P5) hippocampi. This is accompanied by changes in 5hmC signal intensity in different hippocampal regions. Transcriptome analysis of hippocampi and prefrontal cortex, at different developmental stages, indicates that lack of Aag alters gene expression, primarily of genes involved in regulation of response to stress. Across all developmental stages tested aldehyde dehydrogenase 2 (Aldh2) emerged as one of the most prominent genes deregulated in Aag-dependent manner. In line with the changes in hippocampal DNA damage levels and the gene expression, adult Aag-/- mice exhibit altered behavior, evident in decreased anxiety levels determined in the Elevated Zero Maze and increased alternations in the Elevated T Maze tests. Taken together these results suggests that Aag has functions in modulation of genome dynamics during brain development, important for animal behavior.
Assuntos
DNA Glicosilases , Humanos , Camundongos , Animais , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA , Ansiedade/genética , Encéfalo/metabolismo , Expressão Gênica , Mamíferos/genéticaRESUMO
Following DNA damage, cells display complex multi-pathway signaling dynamics that connect cell-cycle arrest and DNA repair in G1, S, or G2/M phase with phenotypic fate decisions made between survival, cell-cycle re-entry and proliferation, permanent cell-cycle arrest, or cell death. How these phenotypic fate decisions are determined remains poorly understood, but must derive from integrating genotoxic stress signals together with inputs from the local microenvironment. To investigate this in a systematic manner, we undertook a quantitative time-resolved cell signaling and phenotypic response study in U2OS cells receiving doxorubicin-induced DNA damage in the presence or absence of TNFα co-treatment; we measured key nodes in a broad set of DNA damage signal transduction pathways along with apoptotic death and cell-cycle regulatory responses. Two relational modeling approaches were then used to identify network-level relationships between signals and cell phenotypic events: a partial least squares regression approach and a complementary new technique which we term 'time-interval stepwise regression.' Taken together, the results from these analysis methods revealed complex, cytokine-modulated inter-relationships among multiple signaling pathways following DNA damage, and identified an unexpected context-dependent role for Erk in both G1/S arrest and apoptotic cell death following treatment with this commonly used clinical chemotherapeutic drug.
Assuntos
Apoptose/genética , Dano ao DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Apoptose/fisiologia , Microambiente Celular/genética , Microambiente Celular/fisiologia , Biologia Computacional/métodos , Dano ao DNA/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Modelos Biológicos , Modelos Teóricos , Transdução de Sinais/genética , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Biologia de Sistemas/métodos , Células Tumorais Cultivadas , Estudos de Validação como AssuntoRESUMO
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
To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.
Assuntos
Dano ao DNA , DNA Glicosilases/química , DNA Glicosilases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Catálise , Cristalografia por Raios X , Adutos de DNA/química , DNA Glicosilases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Humanos , Mutagênese , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Plasmídeos , Conformação ProteicaRESUMO
Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.
Assuntos
Citosina/análogos & derivados , DNA Glicosilases/química , DNA de Neoplasias/química , Proteínas de Neoplasias/química , Domínio Catalítico , Citosina/química , Citosina/metabolismo , Dano ao DNA/fisiologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Humanos , Ligação de Hidrogênio , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Relação Estrutura-AtividadeRESUMO
Alkylation-induced O(6)-methylguanine (O(6)MeG) DNA lesions can be mutagenic or cytotoxic if unrepaired by the O(6)MeG-DNA methyltransferase (Mgmt) protein. O(6)MeG pairs with T during DNA replication, and if the O(6)MeG:T mismatch persists, a G:C to A:T transition mutation is fixed at the next replication cycle. O(6)MeG:T mismatch detection by MutSalpha and MutLalpha leads to apoptotic cell death, but the mechanism by which this occurs has been elusive. To explore how mismatch repair mediates O(6)MeG-dependent apoptosis, we used an Mgmt-null mouse model combined with either the Msh6-null mutant (defective in mismatch recognition) or the Exo1-null mutant (impaired in the excision step of mismatch repair). Mouse embryonic fibroblasts and bone marrow cells derived from Mgmt-null mice were much more alkylation-sensitive than wild type, as expected. However, ablation of either Msh6 or Exo1 function rendered these Mgmt-null cells just as resistant to alkylation-induced cytotoxicity as wild-type cells. Rapidly proliferating tissues in Mgmt-null mice (bone marrow, thymus, and spleen) are extremely sensitive to apoptosis induced by O(6)MeG-producing agents. Here, we show that ablation of either Msh6 or Exo1 function in the Mgmt-null mouse renders these rapidly proliferating tissues alkylation-resistant. However, whereas the Msh6 defect confers total alkylation resistance, the Exo1 defect leads to a variable tissue-specific alkylation resistance phenotype. Our results indicate that Exo1 plays an important role in the induction of apoptosis by unrepaired O(6)MeGs.