RESUMO
Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.
Assuntos
Replicação do DNA , DNA de Cadeia Simples , Humanos , DNA de Cadeia Simples/genética , Replicação do DNA/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Ligação Proteica , Ubiquitinação , Dano ao DNA , Instabilidade Genômica , DNA Helicases/genética , DNA Helicases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Genetic studies in mice and human cancers established BCL11B as a haploinsufficient tumor suppressor gene. Paradoxically, BCL11B is overexpressed in some human cancers where its knockdown is synthetic lethal. We identified the BCL11B protein in a proximity-dependent biotinylation screen performed with the DNA glycosylase NTHL1. In vitro DNA repair assays demonstrated that both BCL11B and a small recombinant BCL11B213-560 protein lacking transcription regulation potential can stimulate the enzymatic activities of two base excision repair (BER) enzymes: NTHL1 and Pol ß. In cells, BCL11B is rapidly recruited to sites of DNA damage caused by laser microirradiation. BCL11B knockdown delays, whereas ectopic expression of BCL11B213-560 accelerates, the repair of oxidative DNA damage. Inactivation of one BCL11B allele in TK6 lymphoblastoid cells causes an increase in spontaneous and radiation-induced mutation rates. In turn, ectopic expression of BCL11B213-560 cooperates with the RAS oncogene in cell transformation by reducing DNA damage and cellular senescence. These findings indicate that BCL11B functions as a BER accessory factor, safeguarding normal cells from acquiring mutations. Paradoxically, it also enables the survival of cancer cells that would otherwise undergo senescence or apoptosis due to oxidative DNA damage resulting from the elevated production of reactive oxygen species.
Assuntos
Reparo por Excisão , Proteínas Repressoras , Animais , Humanos , Camundongos , Dano ao DNA , Reparo do DNA/genética , Genes Supressores de Tumor , Oncogenes , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.
RESUMO
The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.
Assuntos
Homólogo 5 da Proteína Cromobox , Reparo do DNA , Deficiência Intelectual , Reparo de DNA por Recombinação , Transposases , Animais , Homólogo 5 da Proteína Cromobox/genética , Homólogo 5 da Proteína Cromobox/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA , Quebras de DNA de Cadeia Dupla , Humanos , Deficiência Intelectual/genética , Camundongos , Transposases/genética , Transposases/metabolismoRESUMO
DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas Mad2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Fase G2/genética , Células HEK293 , Humanos , Proteínas Mad2/genética , Fase S/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR.
Assuntos
Dano ao DNA , Enzimas Reparadoras do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas Nucleares/fisiologia , Proteína de Replicação A/metabolismo , Ubiquitina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Quinase 1 do Ponto de Checagem , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Fosforilação , Proteínas Quinases/metabolismo , Fatores de Processamento de RNA , Proteína de Replicação A/fisiologia , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Sumoilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/genética , Transporte Proteico , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Dano ao DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Troca , Genes cdc , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Treonina/genéticaRESUMO
RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.
Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA , DNA de Cadeia Simples/genética , Recombinação Homóloga , Proteínas Nucleares/genética , Fatores de Processamento de RNA/genética , Proteína de Replicação A/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fosforilação , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Proteína de Replicação A/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The complete and accurate replication of the genome is a crucial aspect of cell proliferation that is often perturbed during oncogenesis. Replication stress arising from a variety of obstacles to replication fork progression and processivity is an important contributor to genome destabilization. Accordingly, cells mount a complex response to this stress that allows the stabilization and restart of stalled replication forks and enables the full duplication of the genetic material. This response articulates itself on three important platforms, Replication Protein A/RPA-coated single-stranded DNA, the DNA polymerase processivity clamp PCNA and the FANCD2/I Fanconi Anemia complex. On these platforms, the recruitment, activation and release of a variety of genome maintenance factors is regulated by post-translational modifications including mono- and poly-ubiquitylation. Here, we review recent insights into the control of replication fork stability and restart by the ubiquitin system during replication stress with a particular focus on human cells. We highlight the roles of E3 ubiquitin ligases, ubiquitin readers and deubiquitylases that provide the required flexibility at stalled forks to select the optimal restart pathways and rescue genome stability during stressful conditions.
Assuntos
Replicação do DNA , DNA/genética , Anemia de Fanconi/genética , Ubiquitinação , Animais , DNA/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Instabilidade Genômica , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reparo do DNA , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Ciprofloxacina , Quebras de DNA de Cadeia Dupla , DNA de Plantas/metabolismo , Rearranjo Gênico , Dados de Sequência Molecular , Inibidores da Síntese de Ácido Nucleico , Plastídeos/genética , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Illudin S (ILS) is a fungal sesquiterpene secondary metabolite with potent genotoxic and cytotoxic properties. Early genetic studies and more recent genome-wide CRISPR screens showed that Illudin-induced lesions are preferentially repaired by transcription-coupled nucleotide excision repair (TC-NER) with some contribution from post-replication repair pathways. In line with these results, Irofulven, a semi-synthetic ILS analog was recently shown to be particularly effective on cell lines and patient-derived xenografts with impaired NER (e.g. ERCC2/3 mutations), raising hope that ILS-derived molecules may soon enter the clinic. Despite the therapeutic potential of ILS and its analogs, we still lack a global understanding of their mutagenic potential. Here, we characterize the mutational signatures associated with chronic exposure to ILS in human cells. ILS treatment rapidly stalls DNA replication and transcription, leading to the activation of the replication stress response and the accumulation of DNA damage. Novel single and double base substitution signatures as well as a characteristic indel signature indicate that ILS treatment preferentially alkylates purine residues and induces oxidative stress, confirming prior in vitro data. Many mutation contexts exhibit a strong transcriptional strand bias, highlighting the contribution of TC-NER to the repair of ILS lesions. Finally, collateral mutations are also observed in response to ILS, suggesting a contribution of translesion synthesis pathways to ILS tolerance. Accordingly, ILS treatment led to the rapid recruitment of the Y-family DNA polymerase kappa onto chromatin, supporting its preferential use for ILS lesion bypass. Altogether, our work provides the first global assessment of the genomic impact of ILS, demonstrating the contribution of multiple DNA repair pathways to ILS resistance and mutagenicity.
Assuntos
Dano ao DNA , Reparo do DNA , Humanos , Dano ao DNA/genética , Reparo do DNA/genética , Mutagênese/genética , Mutagênicos , Mutação , Transcrição Gênica , Proteína Grupo D do Xeroderma Pigmentoso/genética , Estresse OxidativoRESUMO
As modern agricultural practices increase their use of chemical pesticides, it is inevitable that we will find a number of these xenobiotics within drinking water supplies and disseminated throughout the food chain. A major problem that arises from this pollution is that the effects of most of these pesticides on cellular mechanisms in general, and how they interact with each other and affect human cells are still poorly understood. In this study we make use of cultured human cancer cells to measure by qRT-PCR how pesticides affect gene expression of stress pathways. Immunoblotting studies were performed to monitor protein expression levels and activation of signaling pathways. We make use of immunofluorescence and microscopy to visualize and quantify DNA damage events in those cells. In the current study, we evaluate the potential of a subset of widely used pesticides to activate the dioxin receptor pathway and affect its crosstalk with estrogen receptor signaling. We quantify the impact of these chemicals on the p53-dependent cellular stress response. We find that, not only can the different pesticides activate the dioxin receptor pathway, most of them have better than additive effects on this pathway when combined at low doses. We also show that different pesticides have the ability to trigger crosstalk events that may generate genotoxic estrogen metabolites. Finally, we show that some, but not all of the tested pesticides can induce a p53-dependent stress response. Taken together our results provide evidence that several xenobiotics found within the environment have the potential to interact together to elicit significant effects on cell systems. Our data warrants caution when the toxicity of substances that are assessed simply for individual chemicals, since important biological effects could be observed only in the presence of other compounds, and that even at very low concentrations.
Assuntos
Dioxinas , Praguicidas , Dibenzodioxinas Policloradas , Humanos , Praguicidas/toxicidade , Praguicidas/química , Dioxinas/toxicidade , Receptores de Hidrocarboneto Arílico , Proteína Supressora de Tumor p53/genéticaRESUMO
DNA replication stress (RS) entails the frequent slow down and arrest of replication forks by a variety of conditions that hinder accurate and processive genome duplication. Elevated RS leads to genome instability, replication catastrophe and eventually cell death. RS is particularly prevalent in cancer cells and its exacerbation to unsustainable levels by chemotherapeutic agents remains a cornerstone of cancer treatments. The adverse consequences of RS are normally prevented by the ATR and CHK1 checkpoint kinases that stabilize stressed forks, suppress origin firing and promote cell cycle arrest when replication is perturbed. Specific inhibitors of these kinases have been developed and shown to potentiate RS and cell death in multiple in vitro cancer settings. Ongoing clinical trials are now probing their efficacy against various cancer types, either as single agents or in combination with mainstay chemotherapeutics. Despite their promise as valuable additions to the anti-cancer pharmacopoeia, we still lack a genome-wide view of the potential mutagenicity of these new drugs. To investigate this question, we performed chronic long-term treatments of TP53-depleted human cancer cells with ATR and CHK1 inhibitors (ATRi, AZD6738/ceralasertib and CHK1i, MK8776/SCH-900776). ATR or CHK1 inhibition did not significantly increase the mutational burden of cells, nor generate specific mutational signatures. Indeed, no notable changes in the numbers of base substitutions, short insertions/deletions and larger scale rearrangements were observed despite induction of replication-associated DNA breaks during treatments. Interestingly, ATR inhibition did induce a slight increase in closely-spaced mutations, a feature previously attributed to translesion synthesis DNA polymerases. The results suggest that ATRi and CHK1i do not have substantial mutagenic effects in vitro when used as standalone agents.
Assuntos
Dano ao DNA , Neoplasias , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Replicação do DNA , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismoRESUMO
Maintenance of genome stability is essential for the accurate propagation of genetic information and cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively, little is known about how organelle genomes maintain a stable structure. Here, we report that the plastid-localized Whirly ssDNA-binding proteins are required for plastid genome stability in Arabidopsis. We show that a double KO of the genes AtWhy1 and AtWhy3 leads to the appearance of plants with variegated green/white/yellow leaves, symptomatic of nonfunctional chloroplasts. This variegation is maternally inherited, indicating defects in the plastid genome. Indeed, in all variegated lines examined, reorganized regions of plastid DNA are amplified as circular and/or head-tail concatemers. All amplified regions are delimited by short direct repeats of 10-18 bp, strongly suggesting that these regions result from illegitimate recombination between repeated sequences. This type of recombination occurs frequently in plants lacking both Whirlies, to a lesser extent in single KO plants and rarely in WT individuals. Maize mutants for the ZmWhy1 Whirly protein also show an increase in the frequency of illegitimate recombination. We propose a model where Whirlies contribute to plastid genome stability by protecting against illegitimate repeat-mediated recombination.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , DNA de Cloroplastos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Cloroplastos/genética , Cloroplastos/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase , Ligação Proteica , Zea mays/genética , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
mRNA processing factors are increasingly being recognized as important regulators of genome stability. By preventing and resolving RNA:DNA hybrids that form co-transcriptionally, these proteins help avoid replication-transcription conflicts and thus contribute to genome stability through their normal function in RNA maturation. Some of these factors also have direct roles in the activation of the DNA damage response and in DNA repair. One of the most intriguing cases is that of PRP19, an evolutionarily conserved essential E3 ubiquitin ligase that promotes mRNA splicing, but also participates directly in ATR activation, double-strand break resection and mitosis. Here, we review historical and recent work on PRP19 and its associated proteins, highlighting their multifarious cellular functions as central regulators of spliceosome activity, R-loop homeostasis, DNA damage signaling and repair and cell division. Finally, we discuss open questions that are bound to shed further light on the functions of PRP19-containing complexes in both normal and cancer cells.
RESUMO
The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase ß to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol ß in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.
Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Reparo do DNA , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Linhagem Celular , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Like their nuclear counterpart, the plastid and mitochondrial genomes of plants have to be faithfully replicated and repaired to ensure the normal functioning of the plant. Inability to maintain organelle genome stability results in plastid and/or mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly, deregulation of recombination in organelles results in genomic instability, often accompanied by adverse consequences for plant fitness. The recent identification of four families of proteins that prevent aberrant recombination of organelle DNA sheds much needed mechanistic light on this important process. What comes out of these investigations is a partial portrait of the recombination surveillance machinery in which plants have co-opted some proteins of prokaryotic origin but have also evolved whole new factors to keep their organelle genomes intact. These new features presumably optimized the protection of plastid and mitochondrial genomes against the particular genotoxic stresses they face.
Assuntos
Genoma de Planta/genética , Instabilidade Genômica/genética , Organelas/genética , Plantas/genética , Recombinação Genética/genéticaRESUMO
Aim: To comprehensively characterize the high-density lipoproteins (HDLs) microtranscriptome and to assess whether it is distinct from that of plasma and different between women and men. Methods: RNA was extracted from ultracentrifugation-purified HDLs and plasma from 17 healthy women and men couples, and libraries were sequenced on a HiSeq2500 platform. Results: On average, 310 ± 64 and 355 ± 31 miRNAs were detected (≥1 read per million) in HDLs and plasma, respectively. A total of 62 and 134 miRNAs were over-represented (e.g., miR-150-5p; fold change = 7.52; padj = 5.41 × 10-111) and under-represented (e.g., miR-22-3p; fold change = -5.28; padj = 2.11 × 10-154) in HDLs compared with plasma. These miRNAs were enriched in lipid metabolism and cellular processes-related pathways. Conclusion: HDLs exhibit a sex-independent miRNA profile distinct from that of plasma. These miRNAs may contribute to the HDLs' physiology.
Assuntos
Metabolismo dos Lipídeos/genética , Lipoproteínas HDL/genética , MicroRNAs/genética , Transcriptoma , Adulto , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lipoproteínas HDL/sangue , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , Fatores Sexuais , Adulto JovemRESUMO
BACKGROUND: StWhy1, a member of the plant-specific Whirly single-stranded DNA-binding protein family, was first characterized as a transcription factor involved in the activation of the nuclear PR-10a gene following defense-related stress in potato. In Arabidopsis thaliana, Whirlies have recently been shown to be primarily localized in organelles. Two representatives of the family, AtWhy1 and AtWhy3 are imported into plastids while AtWhy2 localizes to mitochondria. Their function in organelles is currently unknown. RESULTS: To understand the role of mitochondrial Whirlies in higher plants, we produced A. thaliana lines with altered expression of the atwhy2 gene. Organellar DNA immunoprecipitation experiments demonstrated that AtWhy2 binds to mitochondrial DNA. Overexpression of atwhy2 in plants perturbs mitochondrial function by causing a diminution in transcript levels and mtDNA content which translates into a low activity level of respiratory chain complexes containing mtDNA-encoded subunits. This lowered activity of mitochondria yielded plants that were reduced in size and had distorted leaves that exhibited accelerated senescence. Overexpression of atwhy2 also led to early accumulation of senescence marker transcripts in mature leaves. Inactivation of the atwhy2 gene did not affect plant development and had no detectable effect on mitochondrial morphology, activity of respiratory chain complexes, transcription or the amount of mtDNA present. This lack of phenotype upon abrogation of atwhy2 expression suggests the presence of functional homologues of the Whirlies or the activation of compensating mechanisms in mitochondria. CONCLUSION: AtWhy2 is associated with mtDNA and its overexpression results in the production of dysfunctional mitochondria. This report constitutes the first evidence of a function for the Whirlies in organelles. We propose that they could play a role in the regulation of the gene expression machinery of organelles.