RESUMO
Accurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming. Here, we discuss recent advances toward our understanding of the mechanisms that regulate the fine-tuned balance between these different replication stress response pathways. We also discuss the molecular pathways required to fill single-stranded DNA gaps that accumulate throughout the genome after repriming and the biological consequences of using repriming instead of other DNA damage tolerance pathways on genome integrity and cell fitness.
Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica , Animais , DNA de Cadeia Simples/genética , HumanosRESUMO
RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.
Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Origem de Replicação/genética , Linhagem Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/genética , Rad51 Recombinase/genéticaRESUMO
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
Assuntos
Quebras de DNA de Cadeia Simples , DNA Primase/metabolismo , Reparo do DNA , Replicação do DNA , DNA de Neoplasias/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Fase G2 , Enzimas Multifuncionais/metabolismo , Neoplasias/metabolismo , Fase S , Antineoplásicos/farmacologia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Primase/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica , Células HEK293 , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Enzimas Multifuncionais/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
DNA replication forks use multiple mechanisms to deal with replication stress, but how the choice of mechanisms is made is still poorly understood. Here, we show that CARM1 associates with replication forks and reduces fork speed independently of its methyltransferase activity. The speeding of replication forks in CARM1-deficient cells requires RECQ1, which resolves reversed forks, and RAD18, which promotes translesion synthesis. Loss of CARM1 reduces fork reversal and increases single-stranded DNA (ssDNA) gaps but allows cells to tolerate higher replication stress. Mechanistically, CARM1 interacts with PARP1 and promotes PARylation at replication forks. In vitro, CARM1 stimulates PARP1 activity by enhancing its DNA binding and acts jointly with HPF1 to activate PARP1. Thus, by stimulating PARP1, CARM1 slows replication forks and promotes the use of fork reversal in the stress response, revealing that CARM1 and PARP1 function as a regulatory module at forks to control fork speed and the choice of stress response mechanisms.
Assuntos
Quebras de DNA de Cadeia Simples , Replicação do DNA , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Proteína-Arginina N-Metiltransferases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.
Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Núcleo Celular/enzimologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Neoplásico/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Núcleo Celular/genética , DNA Helicases/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Metilação , Neoplasias/genética , Proteínas Nucleares/genética , Estruturas R-Loop , RNA Neoplásico/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Transcrição Gênica , UbiquitinaçãoRESUMO
Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Citidina Desaminase , Dano ao DNA , Replicação do DNA , Humanos , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Instabilidade Genômica , Linhagem Celular Tumoral , ProteínasRESUMO
Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.
Assuntos
DNA Primase/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Primase/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Células HEK293 , Humanos , Enzimas Multifuncionais/fisiologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Enzimas Reparadoras do DNA/genética , Reparo do DNA , Replicação do DNA , Exodesoxirribonucleases/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sinalização do Cálcio/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/química , Cromatina/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.
RESUMO
In a lifetime, a human being synthesizes approximately 2×1016 meters of DNA, a distance that corresponds to 130,000 times the distance between the Earth and the Sun. This daunting task is executed by thousands of replication forks, which progress along the chromosomes and frequently stall when they encounter DNA lesions, unusual DNA structures, RNA polymerases, or tightly-bound protein complexes. To complete DNA synthesis before the onset of mitosis, eukaryotic cells have evolved complex mechanisms to process and restart arrested forks through the coordinated action of multiple nucleases, topoisomerases, and helicases. In this review, we discuss recent advances in understanding the role and regulation of nucleases acting at stalled forks with a focus on the nucleolytic degradation of nascent DNA, a process commonly referred to as fork resection. We also discuss the effects of deregulated fork resection on genomic instability and on the unscheduled activation of the interferon response under replication stress conditions.
Assuntos
DNA Helicases/genética , Replicação do DNA , DNA Topoisomerases/genética , DNA/genética , Endonucleases/genética , Reparo de DNA por Recombinação , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , DNA Helicases/metabolismo , DNA Topoisomerases/metabolismo , Endonucleases/metabolismo , Instabilidade Genômica , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
Replication fork reversal is a rapidly emerging and remarkably frequent mechanism of fork stabilization in response to genotoxic insults. Here, we summarize recent findings that uncover key molecular determinants for reversed fork formation and describe how the homologous recombination factors BRCA1, BRCA2, and RAD51 protect these structures from extended nucleolytic degradation.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Dano ao DNA , Replicação do DNA , DNA/biossíntese , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , DNA/genética , Humanos , Rad51 Recombinase/genéticaRESUMO
DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome. The Primase and DNA-directed Polymerase (PRIMPOL) is a recently discovered enzyme that possesses both primase and polymerase activities. PRIMPOL is emerging as a key player in DNA damage tolerance, particularly in vertebrate and human cells. Here, we review our current understanding of the function of PRIMPOL in DNA damage tolerance by focusing on the structural aspects that define its dual enzymatic activity, as well as on the mechanisms that control its chromatin recruitment and expression levels. We also focus on the latest findings on the mitochondrial and nuclear functions of PRIMPOL and on the impact of loss of these functions on genome stability and cell survival. Defining the function of PRIMPOL in DNA damage tolerance is becoming increasingly important in the context of human disease. In particular, we discuss recent evidence pointing at the PRIMPOL pathway as a novel molecular target to improve cancer cell response to DNA-damaging chemotherapy and as a predictive parameter to stratify patients in personalized cancer therapy.
Assuntos
Dano ao DNA/genética , DNA Primase/genética , DNA Primase/metabolismo , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/genética , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Humanos , Mitocôndrias/metabolismo , Enzimas Multifuncionais/químicaRESUMO
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Assuntos
Enzimas AlkB/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Síndromes de Tricotiodistrofia/genética , Ubiquitina/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilantes/farmacologia , Alquilação , Sequência de Aminoácidos , Adutos de DNA/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Genes Ligados ao Cromossomo X , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , RNA Polimerase II/metabolismo , Splicing de RNA , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologia , UbiquitinaçãoRESUMO
Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.
Assuntos
Replicação do DNA , Lamina Tipo A/metabolismo , Modelos Biológicos , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Animais , Células HEK293 , Humanos , Lamina Tipo A/genética , Camundongos , Camundongos Knockout , Rad51 Recombinase/genética , Proteína de Replicação A/genéticaRESUMO
TAR DNA-binding protein 43 (TDP-43; also known as TARDBP) is an RNA-binding protein whose aggregation is a hallmark of the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 loss increases DNA damage and compromises cell viability, but the actual function of TDP-43 in preventing genome instability remains unclear. Here, we show that loss of TDP-43 increases R-loop formation in a transcription-dependent manner and results in DNA replication stress. TDP-43 nucleic-acid-binding and self-assembly activities are important in inhibiting R-loop accumulation and preserving normal DNA replication. We also found that TDP-43 cytoplasmic aggregation impairs TDP-43 function in R-loop regulation. Furthermore, increased R-loop accumulation and DNA damage is observed in neurons upon loss of TDP-43. Together, our findings indicate that TDP-43 function and normal protein homeostasis are crucial in maintaining genomic stability through a co-transcriptional process that prevents aberrant R-loop accumulation. We propose that the increased R-loop formation and genomic instability associated with TDP-43 loss are linked to the pathogenesis of TDP-43 proteinopathies.This article has an associated First Person interview with the first author of the paper.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Estruturas R-LoopRESUMO
Magnetic materials interfaced with superconductors may reveal new physical phenomena with potential for quantum technologies. The use of molecules as magnetic components has already shown great promise, but the diversity of properties offered by the molecular realm remains largely unexplored. Here we investigate a submonolayer of tetrairon(III) propeller-shaped single molecule magnets deposited on a superconducting lead surface. This material combination reveals a strong influence of the superconductor on the spin dynamics of the single molecule magnet. It is shown that the superconducting transition to the condensate state switches the single molecule magnet from a blocked magnetization state to a resonant quantum tunnelling regime. Our results open perspectives to control single molecule magnetism via superconductors and to use single molecule magnets as local probes of the superconducting state.
RESUMO
OBJECTIVE: Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. METHODS: We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. RESULTS: Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. CONCLUSION: Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/farmacologia , Carcinoma Epitelial do Ovário/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Piridinas/farmacologia , Animais , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/biossíntese , Proteína BRCA1/genética , Benzamidas/administração & dosagem , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Inibidores de Histona Desacetilases/administração & dosagem , Recombinação Homóloga , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Peritoneais/prevenção & controle , Neoplasias Peritoneais/secundário , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Piridinas/administração & dosagem , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pds5 is required for sister chromatid cohesion, and somewhat paradoxically, to remove cohesin from chromosomes. We found that Pds5 plays a critical role during DNA replication that is distinct from its previously known functions. Loss of Pds5 hinders replication fork progression in unperturbed human and mouse cells. Inhibition of MRE11 nuclease activity restores fork progression, suggesting that Pds5 protects forks from MRE11-activity. Loss of Pds5 also leads to double-strand breaks, which are again reduced by MRE11 inhibition. The replication function of Pds5 is independent of its previously reported interaction with BRCA2. Unlike Pds5, BRCA2 protects forks from nucleolytic degradation only in the presence of genotoxic stress. Moreover, our iPOND analysis shows that the loading of Pds5 and other cohesion factors on replication forks is not affected by the BRCA2 status. Pds5 role in DNA replication is shared by the other cohesin-removal factor Wapl, but not by the cohesin complex component Rad21. Interestingly, depletion of Rad21 in a Pds5-deficient background rescues the phenotype observed upon Pds5 depletion alone. These findings support a model where loss of either component of the cohesin releasin complex perturbs cohesin dynamics on replication forks, hindering fork progression and promoting MRE11-dependent fork slowing.
Assuntos
Replicação do DNA/genética , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA , Desoxirribonucleases/genética , Humanos , Troca de Cromátide Irmã/genética , CoesinasRESUMO
The possibility to operate on magnetic materials through the application of electric rather than magnetic fields-promising faster, more compact and energy efficient circuits-continues to spur the investigation of magnetoelectric effects. Symmetry considerations, in particular the lack of an inversion centre, characterize the magnetoelectric effect. In addition, spin-orbit coupling is generally considered necessary to make a spin system sensitive to a charge distribution. However, a magnetoelectric effect not relying on spin-orbit coupling is appealing for spin-based quantum technologies. Here, we report the detection of a magnetoelectric effect that we attribute to an electric field modulation of the magnetic exchange interaction without atomic displacement. The effect is visible in electron paramagnetic resonance absorption of molecular helices under electric field modulation and confirmed by specific symmetry properties and spectral simulation.
Assuntos
Eletricidade , Campos Magnéticos , Compostos Organometálicos/química , Manganês/química , Modelos Moleculares , Conformação MolecularRESUMO
Cancers with deficiencies in homologous recombination-mediated DNA repair (HRR) demonstrate improved clinical outcomes and increased survival. Approximately 50% of high-grade serous ovarian cancers (HGSOC) exhibit homologous recombination deficiency (HRD). HRD can be caused by germline or somatic mutations of genes involved in the HR pathway. Given platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPis) are used in HGSOC, double-strand breaks (DSBs) are common. Unrepaired DSBs are toxic to cells as genomic instability ensues and cells eventually die. Thus, tumor cells with DSBs utilize the high-fidelity HRR as one of the central pathways for repair. In tumors that have HRD, an alternate pathway such as non-homologous end-joining (NHEJ) is used and leads to error-prone repair. To date, methods for clinical detection of homologous recombination deficiency (HRD) are limited to genomic changes of HRR genes and genomic mutation patterns resulting from HRD genes involved in HR-mediated DNA repair. However, these tests detect genomic scars that might not always correlate well with PARP inhibitor or platinum sensitivity in the current state. Therefore, a functional HRD assay should be able to more accurately predict tumor response in real-time. RAD51 foci formation has been used as a functional assay to define HRD and closely correlates with chemotherapy and PARPi sensitivity. The inability to form RAD51 foci is a common feature of HRD. DNA damage can also cause transient slowing or stalling of replication forks defined as replication stress. Replication fork stalling can lead to fork degradation and decreased cell viability if forks do not resume DNA synthesis. Fork degradation has been found to lead to chemosensitivity in BRCA-deficient tumors. To determine this fork degradation phenotype, replication fork/DNA fiber assays are utilized. This review will highlight functional assays for HRD in the context of translating these to real-time clinical assays.