RESUMO
Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.
Assuntos
Autofagia , Dano ao DNA , DNA Helicases , Inflamação , Enzimas Multifuncionais , Nucleotidiltransferases , Estruturas R-Loop , RNA Helicases , Humanos , Autofagia/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiência , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/deficiência , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/deficiência , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Imunidade Inata , Inflamação/patologia , Inflamação/metabolismo , Inflamação/genética , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Fosfoproteínas , RNA Helicases/metabolismo , RNA Helicases/genéticaRESUMO
Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCGâTTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3â5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.
Assuntos
5-Metilcitosina , Citosina , Metilação de DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Citosina/metabolismo , Citosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Reparo do DNA , Dano ao DNA , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , DNA Polimerase iota , DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Replicação do DNA , 8-Hidroxi-2'-Desoxiguanosina/metabolismoRESUMO
Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.
Assuntos
DNA Helicases , DNA Primase , Replicação do DNA , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA , Proteína Homóloga a MRE11 , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Primase/metabolismo , DNA Primase/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dano ao DNA , Ftalazinas/farmacologia , Piperazinas/farmacologia , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genéticaRESUMO
OBJECTIVES: Cancer cells undergo metabolic reprogramming to adapt to high oxidative stress, but little is known about how metabolic remodeling enables gastric cancer cells to survive stress associated with aberrant reactive oxygen species (ROS) production. Here, we aimed to identify the key metabolic enzymes that protect gastric cancer (GC) cells from oxidative stress. METHODS: ROS level was detected by DCFH-DA probes. Multiple cell biological studies were performed to identify the underlying mechanisms. Furthermore, cell-based xenograft and patient-derived xenograft (PDX) model were performed to evaluate the role of MTHFD2 in vivo. RESULTS: We found that overexpression of MTHFD2, but not MTHFD1, is associated with reduced overall and disease-free survival in gastric cancer. In addition, MTHFD2 knockdown reduces the cellular NADPH/NADP+ ratio, colony formation and mitochondrial function, increases cellular ROS and cleaved PARP levels and induces in cell death under hypoxia, a hallmark of solid cancers and a common inducer of oxidative stress. Moreover, genetic or pharmacological inhibition of MTHFD2 reduces tumor burden in both tumor cell lines and patient-derived xenograft-based models. DISCUSSION: our study highlights the crucial role of MTHFD2 in redox regulation and tumor progression, demonstrating the therapeutic potential of targeting MTHFD2.
Assuntos
Progressão da Doença , Homeostase , Metilenotetra-Hidrofolato Desidrogenase (NADP) , Estresse Oxidativo , Neoplasias Gástricas , Animais , Humanos , Camundongos , Aminoidrolases/metabolismo , Aminoidrolases/genética , Linhagem Celular Tumoral , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.
Assuntos
DNA Helicases , Enzimas Multifuncionais , RNA Helicases , RNA não Traduzido , Humanos , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Dano ao DNA , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Agregados Proteicos , Proteostase , Estruturas R-Loop/genética , RNA Helicases/metabolismo , RNA Helicases/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
PrimPol is a human DNA primase-polymerase which restarts DNA synthesis beyond DNA lesions and non-B DNA structures blocking replication. Disfunction of PrimPol in cells leads to slowing of DNA replication rates in mitochondria and nucleus, accumulation of chromosome aberrations, cell cycle delay, and elevated sensitivity to DNA-damaging agents. A defective PrimPol has been suggested to be associated with the development of ophthalmic diseases, elevated mitochondrial toxicity of antiviral drugs and increased cell resistance to chemotherapy. Here, we describe a rare missense PrimPol variant V102A with altered biochemical properties identified in patients suffering from ovarian and cervical cancer. The Val102 to Ala substitution dramatically reduced both the primase and DNA polymerase activities of PrimPol as well as specifically decreased its ability to incorporate ribonucleotides. Structural analysis indicates that the V102A substitution can destabilize the hydrophobic pocket adjacent to the active site, affecting dNTP binding and catalysis.
Assuntos
DNA Primase , DNA Polimerase Dirigida por DNA , Enzimas Multifuncionais , Mutação de Sentido Incorreto , Neoplasias Ovarianas , Neoplasias do Colo do Útero , Feminino , Humanos , Substituição de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , DNA Primase/metabolismo , DNA Primase/química , DNA Primase/genética , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/química , Modelos Moleculares , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/química , Conformação Proteica , Neoplasias do Colo do Útero/genética , Neoplasias Ovarianas/genéticaRESUMO
Ovarian cancer (OC) is a deadliest gynecological cancer with the highest mortality rate. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a crucial tumor-promoting factor, is over-expressed in several malignancies including OC. The present study aimed to explore the role and mechanisms of MTHFD2 in OC malignant progression. Thus, cell proliferation, cycling, apoptosis, migration, and invasion were evaluated by CCK-8 assay, EdU assay, flow cytometry, wound healing, transwell assay and western blotting. Additionally, glycolysis was assessed by measuring the level of glucose and lactate production, as well as the expressions of GLUT1, HK2 and PKM2. Then the expression of ferroptosis-related proteins and ERK signaling was detected using western blotting. Ferroptosis was detected through the measurement of iron level, GSH, MDA and ROS activities. The results revealed that MTHFD2 was highly expressed in OC cells. Besides, interference with MTHFD2 induced ferroptosis, promoted ROS accumulation, destroyed mitochondrial function, reduced ATP content and inhibited glycolysis in OC cells. Subsequently, we further found that interference with MTHFD2 affected mitochondrial function and glycolysis in OC cells through ERK signaling. Moreover, interference with MTHFD2 affected ferroptosis to inhibit the malignant progression of OC cells. Collectively, our present study disclosed that interference with MTHFD2 induced ferroptosis in OC to inhibit tumor malignant progression through regulating ERK signaling.
Assuntos
Ferroptose , Sistema de Sinalização das MAP Quinases , Metilenotetra-Hidrofolato Desidrogenase (NADP) , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/genética , Ferroptose/fisiologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/metabolismo , Linhagem Celular Tumoral , Aminoidrolases/metabolismo , Aminoidrolases/genética , Progressão da Doença , CamundongosRESUMO
DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21 , DNA Primase , Replicação do DNA , DNA Polimerase Dirigida por DNA , Enzimas Multifuncionais , Proteínas de Ligação a Telômeros , Raios Ultravioleta , Humanos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , DNA Primase/metabolismo , DNA Primase/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteínas de Ligação a Telômeros/genética , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Dano ao DNARESUMO
It is well-established that, through canonical functions in transcription and DNA repair, the tumor suppressor p53 plays a central role in safeguarding cells from the consequences of DNA damage. Recent data retrieved in tumor and stem cells demonstrated that p53 also carries out non-canonical functions when interacting with the translesion synthesis (TLS) polymerase iota (POLι) at DNA replication forks. This protein complex triggers a DNA damage tolerance (DDT) mechanism controlling the DNA replication rate. Given that the levels of p53 trigger non-binary rheostat-like functions in response to stress or during differentiation, we explore the relevance of the p53 levels for its DDT functions at the fork. We show that subtle changes in p53 levels modulate the contribution of some DDT factors including POLι, POLη, POLζ, REV1, PCNA, PRIMPOL, HLTF and ZRANB3 to the DNA replication rate. Our results suggest that the levels of p53 are central to coordinate the balance between DDT pathways including (i) fork-deceleration by the ZRANB3-mediated fork reversal factor, (ii) POLι-p53-mediated fork-slowing, (iii) POLι- and POLη-mediated TLS and (iv) PRIMPOL-mediated fork-acceleration. Collectively, our study reveals the relevance of p53 protein levels for the DDT pathway choice in replicating cells.
Assuntos
Dano ao DNA , DNA Polimerase iota , Replicação do DNA , DNA Polimerase Dirigida por DNA , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Reparo do DNA , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , DNA Primase/metabolismo , DNA Primase/genética , Tolerância ao Dano no DNARESUMO
Translesion DNA synthesis (TLS) is a DNA damage tolerance pathway utilized by cells to overcome lesions encountered throughout DNA replication. During replication stress, cancer cells show increased dependency on TLS proteins for cellular survival and chemoresistance. TLS proteins have been described to be involved in various DNA repair pathways. One of the major emerging roles of TLS is single-stranded DNA (ssDNA) gap-filling, primarily after the repriming activity of PrimPol upon encountering a lesion. Conversely, suppression of ssDNA gap accumulation by TLS is considered to represent a mechanism for cancer cells to evade the toxicity of chemotherapeutic agents, specifically in BRCA-deficient cells. Thus, TLS inhibition is emerging as a potential treatment regimen for DNA repair-deficient tumors.
Assuntos
DNA Primase , Reparo do DNA , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA , Enzimas Multifuncionais , Síntese de DNA Translesão , Dano ao DNA , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Animais , DNA Primase/metabolismo , Enzimas Multifuncionais/metabolismoRESUMO
The primase/polymerase PRIMPOL restarts DNA synthesis when replication is arrested by template impediments. However, we do not have a comprehensive view of how PRIMPOL-dependent repriming integrates with the main pathways of damage tolerance, REV1-dependent 'on-the-fly' lesion bypass at the fork and PCNA ubiquitination-dependent post-replicative gap filling. Guided by genome-wide CRISPR/Cas9 screens to survey the genetic interactions of PRIMPOL in a non-transformed and p53-proficient human cell line, we find that PRIMPOL is needed for cell survival following loss of the Y-family polymerases REV1 and POLη in a lesion-dependent manner, while it plays a broader role in promoting survival of cells lacking PCNA K164-dependent post-replicative gap filling. Thus, while REV1- and PCNA K164R-bypass provide two layers of protection to ensure effective damage tolerance, PRIMPOL is required to maximise the effectiveness of the interaction between them. We propose this is through the restriction of post-replicative gap length provided by PRIMPOL-dependent repriming.
Assuntos
Dano ao DNA , DNA Primase , DNA Polimerase Dirigida por DNA , Humanos , DNA Primase/genética , DNA Primase/metabolismo , Replicação do DNA , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , DNA Polimerase Dirigida por DNA/metabolismoRESUMO
The RNA/DNA helicase senataxin (SETX) has been involved in multiple crucial processes related to genome expression and integrity such us transcription termination, the regulation of transcription-replication conflicts and the resolution of R-loops. SETX has been the focus of numerous studies since the discovery that mutations in its coding gene are the root cause of two different neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and a juvenile form of Amyotrophic Lateral Sclerosis (ALS4). A plethora of cellular phenotypes have been described as the result of SETX deficiency, yet the precise molecular function of SETX as well as the molecular pathways leading from SETX mutations to AOA2 and ALS4 pathologies have remained unclear. However, recent data have shed light onto the biochemical activities and biological roles of SETX, thus providing new clues to understand the molecular consequences of SETX mutation. In this review we summarize near two decades of scientific effort to elucidate SETX function, we discuss strengths and limitations of the approaches and models used thus far to investigate SETX-associated diseases and suggest new possible research avenues for the study of AOA2 and ALS4 pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Humanos , RNA Helicases/genética , RNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Doenças Neurodegenerativas/genética , Transcrição Gênica , Mutação , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , RNARESUMO
Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.
Assuntos
Adenocarcinoma de Pulmão , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Células A549 , Cisplatino/farmacologia , Peróxido de Hidrogênio/farmacologia , Replicação do DNA , Dano ao DNA , Adenocarcinoma de Pulmão/genética , DNA Primase/genética , DNA Primase/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.
Assuntos
Domínio Catalítico , Replicação do DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Primase/metabolismo , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , NucleotídeosRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive, uncurable neurodegenerative disorder characterized by the degradation of motor neurons leading to muscle impairment, failure, and death. Senataxin, encoded by the SETX gene, is a human helicase protein whose mutations have been linked with ALS onset, particularly in its juvenile ALS4 form. Using senataxin's yeast homolog Sen1 as a model for study, it is suggested that senataxin's N-terminus interacts with RNA polymerase II, whilst its C-terminus engages in helicase activity. Senataxin is heavily involved in transcription regulation, termination, and R-loop resolution, enabled by recruitment and interactions with enzymes such as ubiquitin protein ligase SAN1 and ribonuclease H (RNase H). Senataxin also engages in DNA damage response (DDR), primarily interacting with the exosome subunit Rrp45. The Sen1 mutation E1597K, alongside the L389S and R2136H gain-of-function mutations to senataxin, is shown to cause negative structural and thus functional effects to the protein, thus contributing to a disruption in WT functions, motor neuron (MN) degeneration, and the manifestation of ALS clinical symptoms. This review corroborates and summarizes published papers concerning the structure and function of senataxin as well as the effects of their mutations in ALS pathology in order to compile current knowledge and provide a reference for future research. The findings compiled in this review are indicative of the experimental and therapeutic potential of senataxin and its mutations as a target in future ALS treatment/cure discovery, with some potential therapeutic routes also being discussed in the review.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Neurônios Motores/metabolismo , Regulação da Expressão Gênica , Mutação , DNA Helicases/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases.
Assuntos
DNA Primase , DNA Polimerase Dirigida por DNA , Humanos , DNA Primase/genética , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Replicação do DNA , DNA/metabolismo , Dano ao DNA , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
Studying the straw lignocellulose strengthening mechanism during simultaneous degradation has important practical significance for improving resource utilization and reducing environmental pollution. In this paper, the degradation ability of four straw lignocellulose-degrading enzymes was evaluated by molecular docking and molecular dynamics. Using the significantly binds to straw lignocellulose-degrading enzyme as a template, a multifunctional lignocellulose-degrading enzyme 3CBH-1KS5-4XQD-1B85 was constructed based on amino acid recombination and homologous modeling. Five efficient degrading enzymes (3CBH-1, 3CBH-2, 3CBH-3, 3CBH-4, and 3CBH-5) were designed by site-directed mutagenesis of 3CBH-1KS5-4XQD-1B85 amino acid at position 346. Molecular dynamics showed that the degradation ability of 3CBH-1 was significant and it was 1.45 times higher than 3CBH-1KS5-4XQD-1B85. Moreover, the mechanism of enhanced degradability and the stability of the enzymes were explored. With the aid of Taguchi experiments, the suitable external environment for degrading straw was determined. In the presence of inhibitors (organic acids and phenolic compounds), the binding energy of 3CBH-1 (238.46 ± 30.96 kJ/mol) is 36.42% higher than that of 3CBH-1KS5-4XQD-1B85 (174.79 ± 20.35 kJ/mol) without external environmental stimulation. Based on homology modeling, this paper constructed a site-directed mutagenesis scheme of multifunctional enzymes, and the aim was to obtain multifunctional and efficient straw lignocellulose-degrading enzymes through protein engineering, which provided a feasible scheme for straw biodegradation.
Assuntos
Simulação de Dinâmica Molecular , Enzimas Multifuncionais , Enzimas Multifuncionais/metabolismo , Simulação de Acoplamento Molecular , Lignina/metabolismo , AminoácidosRESUMO
Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different translesion DNA synthesis (TLS) polymerases by the use of tools for their depletion, we identified a unique role of Pol ι in regulating such a pathway choice. Pol ι deficiency unleashes PrimPol-dependent repriming, which accelerates DNA replication in a pathway that is epistatic with ZRANB3 knockdown. In Pol ι-depleted cells, the excess participation of PrimPol in nascent DNA elongation reduces replication stress signals, but thereby also checkpoint activation in S phase, triggering chromosome instability in M phase. This TLS-independent function of Pol ι requires its PCNA-interacting but not its polymerase domain. Our findings unravel an unanticipated role of Pol ι in protecting the genome stability of cells from detrimental changes in DNA replication dynamics caused by PrimPol.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Dano ao DNA , Instabilidade Cromossômica , DNA Primase/genética , DNA Primase/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismoRESUMO
Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.
Assuntos
DNA Helicases , RNA Helicases , Terminação da Transcrição Genética , Humanos , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Doenças Neurodegenerativas , Estruturas R-Loop , RNA Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Amyotrophic lateral sclerosis (ALS) is regarded as a fatal neurodegenerative disease that is featured by progressive damage of the upper and lower motor neurons. To date, over 45 genes have been found to be connected with ALS pathology. The aim of this work was to computationally identify unique sets of protein hydrolysate peptides that could serve as therapeutic agents against ALS. Computational methods which include target prediction, protein-protein interaction, and peptide-protein molecular docking were used. The results showed that the network of critical ALS-associated genes consists of ATG16L2, SCFD1, VAC15, VEGFA, KEAP1, KIF5A, FIG4, TUBA4A, SIGMAR1, SETX, ANXA11, HNRNPL, NEK1, C9orf72, VCP, RPSA, ATP5B, and SOD1 together with predicted kinases such as AKT1, CDK4, DNAPK, MAPK14, and ERK2 in addition to transcription factors such as MYC, RELA, ZMIZ1, EGR1, TRIM28, and FOXA2. The identified molecular targets of the peptides that support multi-metabolic components in ALS pathogenesis include cyclooxygenase-2, angiotensin I-converting enzyme, dipeptidyl peptidase IV, X-linked inhibitor of apoptosis protein 3, and endothelin receptor ET-A. Overall, the results showed that AGL, APL, AVK, IIW, PVI, and VAY peptides are promising candidates for further study. Future work would be needed to validate the therapeutic properties of these hydrolysate peptides by in vitro and in vivo approaches.