RESUMO
Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event. Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ.
Assuntos
Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Lisina/química , Antígenos Nucleares/química , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Metilação de DNA , Enzimas de Restrição do DNA/farmacologia , Proteínas de Ligação a DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Dimerização , Histona-Lisina N-Metiltransferase/química , Humanos , Autoantígeno Ku , Modelos Teóricos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Saccharomyces cerevisiae/farmacologia , Fatores de TempoRESUMO
Background: The integration of Artificial Intelligence (AI) with synthetic biology is driving unprecedented progress in both fields. However, this integration introduces complex biosecurity challenges. Addressing these concerns, this article proposes a specialized biosecurity risk assessment process designed to evaluate the incorporation of AI in synthetic biology. Methods: A set of tailored tools and methodology was developed for conducting biosecurity risk assessments of AI language models used for synthetic biology. These resources were developed to guide risk management professionals through a systematic process of identifying, evaluating, and mitigating potential risks. Results: The tools and methodology provided offer a structured approach to risk assessment, enabling risk management professionals to comprehensively analyze the biosecurity implications of AI applications in synthetic biology. They facilitate the identification of potential risks and the development of effective mitigation strategies. An example of a risk assessment performed on the large language model "ChatGPT 4.0" is provided here. Conclusion: AI's role in synthetic biology is rapidly expanding; thus, establishing proactive and secure practices is crucial. The biosecurity risk assessment tools and methodology presented here are the first provided in the literature and will be instrumental steps toward the responsible integration of AI in synthetic biology. By adopting these resources, the biorisk management community can effectively navigate and manage the biosecurity challenges posed by AI, ensuring its responsible and secure application in the field of synthetic biology.
RESUMO
Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 µM) and thonzonium bromide (EC(50) = 69 µM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 µM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.
Assuntos
Biguanidas/farmacologia , Inibidores Enzimáticos/farmacologia , Força Próton-Motriz/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Candida albicans/enzimologia , Candida albicans/genética , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Membranas Intracelulares/enzimologia , Mutação , Estrutura Terciária de Proteína , Força Próton-Motriz/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/enzimologia , Vacúolos/genéticaRESUMO
Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9-HUS1-RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.
Assuntos
Reparo do DNA , Replicação do DNA , Histona-Lisina N-Metiltransferase/fisiologia , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/isolamento & purificação , Proliferação de Células , Sobrevivência Celular , DNA Topoisomerases Tipo II/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Imunoprecipitação , Antígeno Nuclear de Célula em Proliferação/isolamento & purificação , Rad51 Recombinase/análiseRESUMO
PURPOSE OF REVIEW: This review highlights recent findings about the known DNA repair machinery, its impact on chromosomal translocation mechanisms and their relevance to leukemia in the clinic. RECENT FINDINGS: Chromosomal translocations regulate the behavior of leukemia. They not only predict outcome but they define therapy. There is a great deal of knowledge on the products of leukemic translocations, yet little is known about the mechanism by which those translocations occur. Given the large number of DNA double-strand breaks that occur during normal progression through the cell cycle, especially from V(D)J recombination, stalled replication forks or failed decatenation, it is surprising that leukemogenic translocations do not occur more frequently. Fortunately, hematopoietic cells have sophisticated repair mechanisms to suppress such translocations. When these defenses fail leukemia becomes far more common, as seen in inherited deficiencies of DNA repair. Analyzing translocation sequences in cellular and animal models, and in human leukemias, has yielded new insights into the mechanisms of leukemogenic translocations. SUMMARY: New data from animal models suggest a two hit origin of leukemic translocations, where there must be both a defect in DNA double-strand break repair and a subsequent failure of cell cycle arrest for leukemogenesis.
Assuntos
Leucemia/genética , Translocação Genética , Ciclo Celular , Reparo do DNA/genética , Humanos , Leucemia/etiologiaRESUMO
BACKGROUND: Here we describe superparamagnetic relaxometry (SPMR), a technology that utilizes highly sensitive magnetic sensors and superparamagnetic nanoparticles for cancer detection. Using SPMR, we sensitively and specifically detect nanoparticles conjugated to biomarkers for various types of cancer. SPMR offers high contrast in vivo, as there is no superparamagnetic background, and bones and tissue are transparent to the magnetic fields. METHODS: In SPMR measurements, a brief magnetizing pulse is used to align superparamagnetic nanoparticles of a discrete size. Following the pulse, an array of superconducting quantum interference detectors (SQUID) sensors detect the decaying magnetization field. NP size is chosen so that, when bound, the induced field decays in seconds. They are functionalized with specific biomarkers and incubated with cancer cells in vitro to determine specificity and cell binding. For in vivo experiments, functionalized NPs are injected into mice with xenograft tumors, and field maps are generated to localize tumor sites. RESULTS: Superparamagnetic NPs developed here have small size dispersion. Cell incubation studies measure specificity for different cell lines and antibodies with very high contrast. In vivo animal measurements verify SPMR localization of tumors. Our results indicate that SPMR possesses sensitivity more than 2 orders of magnitude better than previously reported.
Assuntos
Biomarcadores Tumorais/análise , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Nanopartículas de Magnetita , Neoplasias Experimentais/química , Neoplasias Experimentais/diagnóstico por imagem , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Nus , Camundongos SCID , Imagem Molecular/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and approximately 4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células CHO , Proteínas de Ciclo Celular/antagonistas & inibidores , Cricetinae , Cricetulus , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Ativação Enzimática/efeitos da radiação , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos da radiação , Fosfotreonina/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Radiação Ionizante , Recombinação Genética/genética , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1(XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.