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1.
Nature ; 591(7850): 477-481, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33627873

RESUMO

Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function1. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown2. Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation5 mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I-MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Mitocondrial/imunologia , Imunidade Inata/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Células Cultivadas , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Mitocondrial/efeitos da radiação , Humanos , Mitocôndrias/imunologia , Mitocôndrias/efeitos da radiação , Comunicação Parácrina , Radiação Ionizante , Transcrição Gênica , Ubiquitina-Proteína Ligases/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
2.
PLoS One ; 14(4): e0214552, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30943245

RESUMO

Though it is an essential process, transcription can be a source of genomic instability. For instance, it may generate RNA:DNA hybrids as the nascent transcript hybridizes with the complementary DNA template. These hybrids, called R-loops, act as a major cause of replication fork stalling and DNA breaks. In this study, we show that lowering transcription and R-loop levels in plastids of Arabidopsis thaliana reduces DNA rearrangements and mitigates plastid genome instability phenotypes. This effect can be observed on a genome-wide scale, as the loss of the plastid sigma transcription factor SIG6 prevents DNA rearrangements by favoring conservative repair in the presence of ciprofloxacin-induced DNA damage or in the absence of plastid genome maintenance actors such as WHY1/WHY3, RECA1 and POLIB. Additionally, resolving R-loops by the expression of a plastid-targeted exogenous RNAse H1 produces similar results. We also show that highly-transcribed genes are more susceptible to DNA rearrangements, as increased transcription of the psbD operon by SIG5 correlates with more locus-specific rearrangements. The effect of transcription is not specific to Sigma factors, as decreased global transcription levels by mutation of heat-stress-induced factor HSP21, mutation of nuclear-encoded polymerase RPOTp, or treatment with transcription-inhibitor rifampicin all prevent the formation of plastid genome rearrangements, especially under induced DNA damage conditions.


Assuntos
Arabidopsis/genética , Dano ao DNA , Reparo do DNA , Regulação da Expressão Gênica de Plantas , Genoma de Cloroplastos , Instabilidade Genômica , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Clorofila/química , Cloroplastos/genética , Primers do DNA/genética , Replicação do DNA , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Genoma de Planta , Mutação , Fenótipo , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Recombinases/genética , Rifampina/farmacologia , Análise de Sequência de DNA , Transcrição Gênica
3.
J Cell Biol ; 216(8): 2355-2371, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28637749

RESUMO

Telomerase can generate a novel telomere at DNA double-strand breaks (DSBs), an event called de novo telomere addition. How this activity is suppressed remains unclear. Combining single-molecule imaging and deep sequencing, we show that the budding yeast telomerase RNA (TLC1 RNA) is spatially segregated to the nucleolus and excluded from sites of DNA repair in a cell cycle-dependent manner. Although TLC1 RNA accumulates in the nucleoplasm in G1/S, Pif1 activity promotes TLC1 RNA localization in the nucleolus in G2/M. In the presence of DSBs, TLC1 RNA remains nucleolar in most G2/M cells but accumulates in the nucleoplasm and colocalizes with DSBs in rad52Δ cells, leading to de novo telomere additions. Nucleoplasmic accumulation of TLC1 RNA depends on Cdc13 localization at DSBs and on the SUMO ligase Siz1, which is required for de novo telomere addition in rad52Δ cells. This study reveals novel roles for Pif1, Rad52, and Siz1-dependent sumoylation in the spatial exclusion of telomerase from sites of DNA repair.


Assuntos
Ciclo Celular , Nucléolo Celular/enzimologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/metabolismo , RNA Fúngico/metabolismo , RNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Telomerase/metabolismo , Telômero/metabolismo , Transporte Ativo do Núcleo Celular , Bleomicina/toxicidade , Ciclo Celular/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/efeitos dos fármacos , DNA Fúngico/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , RNA Fúngico/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula , Sumoilação , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
4.
Bioessays ; 37(10): 1086-94, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26222836

RESUMO

In the organelles of plants and mammals, recent evidence suggests that genomic instability stems in large part from template switching events taking place during DNA replication. Although more than one mechanism may be responsible for this, some similarities exist between the different proposed models. These can be separated into two main categories, depending on whether they involve a single-strand-switching or a reciprocal-strand-switching event. Single-strand-switching events lead to intermediates containing Y junctions, whereas reciprocal-strand-switching creates Holliday junctions. Common features in all the described models include replication stress, fork stalling and the presence of inverted repeats, but no single element appears to be required in all cases. We review the field, and examine the ideas that several mechanisms may take place in any given genome, and that the presence of palindromes or inverted repeats in certain regions may favor specific rearrangements.


Assuntos
Replicação do DNA/fisiologia , Instabilidade Genômica/genética , Organelas/genética , Inversão de Sequência/fisiologia , Animais , Humanos , Recombinação Genética , Moldes Genéticos
5.
Genome Res ; 25(5): 645-54, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25800675

RESUMO

Failure to maintain organelle genome stability has been linked to numerous phenotypes, including variegation and cytosolic male sterility (CMS) in plants, as well as cancer and neurodegenerative diseases in mammals. Here we describe a next-generation sequencing approach that precisely maps and characterizes organelle DNA rearrangements in a single genome-wide experiment. In addition to displaying global portraits of genomic instability, it surprisingly unveiled an abundance of short-range rearrangements in Arabidopsis thaliana and human organelles. Among these, short-range U-turn-like inversions reach 25% of total rearrangements in wild-type Arabidopsis plastids and 60% in human mitochondria. Furthermore, we show that replication stress correlates with the accumulation of this type of rearrangement, suggesting that U-turn-like rearrangements could be the outcome of a replication-dependent mechanism. We also show that U-turn-like rearrangements are mostly generated using microhomologies and are repressed in plastids by Whirly proteins WHY1 and WHY3. A synergistic interaction is also observed between the genes for the plastid DNA recombinase RECA1 and those encoding plastid Whirly proteins, and the triple mutant why1why3reca1 accumulates almost 60 times the WT levels of U-turn-like rearrangements. We thus propose that the process leading to U-turn-like rearrangements may constitute a RecA-independent mechanism to restart stalled forks. Our results reveal that short-range rearrangements, and especially U-turn-like rearrangements, are a major factor of genomic instability in organelles, and this raises the question of whether they could have been underestimated in diseases associated with mitochondrial dysfunction.


Assuntos
Arabidopsis/genética , DNA de Cloroplastos/genética , DNA Mitocondrial/genética , Rearranjo Gênico , Genoma Humano , Genoma de Planta , Instabilidade Genômica , Proteínas de Arabidopsis/genética , Ligação Genética , Humanos , Recombinação Genética
6.
Structure ; 23(1): 126-138, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25497731

RESUMO

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Proteínas Correpressoras , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Chaperonas Moleculares , Dados de Sequência Molecular , Fosforilação , Proteína da Leucemia Promielocítica , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
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