RESUMEN
Replication stress and DNA damage stall replication forks and impede genome synthesis. During S phase, damage tolerance pathways allow lesion bypass to ensure efficient genome duplication. One such pathway is repriming, mediated by Primase-Polymerase (PrimPol) in human cells. However, the mechanisms by which PrimPol is regulated are poorly understood. Here, we demonstrate that PrimPol is phosphorylated by Polo-like kinase 1 (PLK1) at a conserved residue between PrimPol's RPA binding motifs. This phosphorylation is differentially modified throughout the cell cycle, which prevents aberrant recruitment of PrimPol to chromatin. Phosphorylation can also be delayed and reversed in response to replication stress. The absence of PLK1-dependent regulation of PrimPol induces phenotypes including chromosome breaks, micronuclei, and decreased survival after treatment with camptothecin, olaparib, and UV-C. Together, these findings establish that deregulated repriming leads to genomic instability, highlighting the importance of regulating this damage tolerance pathway following fork stalling and throughout the cell cycle.
RESUMEN
In light of accumulating evidence suggestive of cell type-specific vulnerabilities as a result of normal aging processes that adversely affect the brain, as well as age-related neurodegenerative disorders such as Parkinson's disease (PD), the current chapter highlights how we study mitochondrial DNA (mtDNA) changes at a single-cell level. In particular, we comment on increasing questioning of the narrow neurocentric view of such pathologies, where microglia and astrocytes have traditionally been considered bystanders rather than players in related pathological processes. Here we review the contribution made by single-cell mtDNA alterations towards neuronal vulnerability seen in neurodegenerative disorders, focusing on PD as a prominent example. In addition, we give an overview of methodologies that support such experimental investigations. In considering the significant advances that have been made in recent times for developing mitochondria-specific therapies, investigations to account for cell type-specific mitochondrial patterns and how these are altered by disease hold promise for delivering more effective disease-modifying therapeutics.
Asunto(s)
Encéfalo/patología , ADN Mitocondrial/análisis , ADN Mitocondrial/genética , Enfermedades Neurodegenerativas/patología , Análisis de la Célula Individual/métodos , Envejecimiento/genética , Humanos , Enfermedades Neurodegenerativas/genética , Enfermedad de Parkinson/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.
Asunto(s)
Aductos de ADN/genética , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Recombinación Homóloga/fisiología , Enzimas Multifuncionales/metabolismo , 4-Nitroquinolina-1-Óxido/toxicidad , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/metabolismo , Benzo(a)Antracenos/administración & dosificación , Benzo(a)Antracenos/toxicidad , Línea Celular , Aductos de ADN/metabolismo , ADN Primasa/genética , ADN de Cadena Simple , ADN Polimerasa Dirigida por ADN/genética , Humanos , Enzimas Multifuncionales/genética , Quinolonas/toxicidad , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Imagen Individual de Molécula , Intercambio de Cromátides HermanasRESUMEN
Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation frequency, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication. Deletion of PrimPol in XPV cells, lacking functional polymerase Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.
Asunto(s)
Núcleo Celular/efectos de la radiación , ADN Primasa/genética , Replicación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/genética , ADN/genética , Mitocondrias/efectos de la radiación , Enzimas Multifuncionales/genética , 4-Nitroquinolina-1-Óxido/farmacología , Bleomicina/farmacología , Línea Celular Transformada , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Cisplatino/farmacología , ADN/efectos de los fármacos , ADN/metabolismo , ADN Primasa/deficiencia , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/deficiencia , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Eliminación de Gen , Humanos , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Enzimas Multifuncionales/deficiencia , Mutágenos/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/efectos de la radiación , Quinolonas/farmacología , Intercambio de Cromátides Hermanas/efectos de los fármacos , Intercambio de Cromátides Hermanas/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
Alzheimer's disease (AD) is the most common form of dementia and is distinguished from other dementias by observation of extracellular Amyloid-ß (Aß) plaques and intracellular neurofibrillary tangles, comprised of fibrils of Aß and tau protein, respectively. At early stages, AD is characterized by minimal neurodegeneration, oxidative stress, nucleolar stress, and altered protein synthesis machinery. It is generally believed that Aß oligomers are the neurotoxic species and their levels in the AD brain correlate with the severity of dementia suggesting that they play a critical role in the pathogenesis of the disease. Here, we show that the incubation of differentiated human neuroblastoma cells (SHSY5Y) with freshly prepared Aß42 oligomers initially resulted in oxidative stress and subtle nucleolar stress in the absence of DNA damage or cell death. The presence of exogenous Aß oligomers resulted in altered nuclear tau levels as well as phosphorylation state, leading to altered distribution of nucleolar tau associated with nucleolar stress. These markers of cellular dysfunction worsen over time alongside a reduction in ribosomal RNA synthesis and processing, a decrease in global level of newly synthesized RNA and reduced protein synthesis. The interplay between Aß and tau in AD remains intriguing and Aß toxicity has been linked to tau phosphorylation and changes in localization. These findings provide evidence for the involvement of Aß42 effects on nucleolar tau and protein synthesis machinery dysfunction in cultured cells. Protein synthesis dysfunction is observed in mild cognitive impairment and early AD in the absence of significant neuronal death.
RESUMEN
Tau is known for its pathological role in neurodegenerative diseases, including Alzheimer's disease (AD) and other tauopathies. Tau is found in many subcellular compartments such as the cytosol and the nucleus. Although its normal role in microtubule binding is well established, its nuclear role is still unclear. Here, we reveal that tau localises to the nucleolus in undifferentiated and differentiated neuroblastoma cells (SHSY5Y), where it associates with TIP5, a key player in heterochromatin stability and ribosomal DNA (rDNA) transcriptional repression. Immunogold labelling on human brain sample confirms the physiological relevance of this finding by showing tau within the nucleolus colocalises with TIP5. Depletion of tau results in an increase in rDNA transcription with an associated decrease in heterochromatin and DNA methylation, suggesting that under normal conditions tau is involved in silencing of the rDNA. Cellular stress induced by glutamate causes nucleolar stress associated with the redistribution of nucleolar non-phosphorylated tau, in a similar manner to fibrillarin, and nuclear upsurge of phosphorylated tau (Thr231) which doesn't colocalise with fibrillarin or nucleolar tau. This suggests that stress may impact on different nuclear tau species. In addition to involvement in rDNA transcription, nucleolar non-phosphorylated tau also undergoes stress-induced redistribution similar to many nucleolar proteins.
Asunto(s)
Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ácido Glutámico/farmacología , Proteínas tau/metabolismo , Encéfalo/metabolismo , Encéfalo/ultraestructura , Diferenciación Celular/fisiología , Línea Celular Tumoral , Nucléolo Celular/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/ultraestructura , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Heterocromatina/fisiología , Histonas/metabolismo , Humanos , Inmunoprecipitación , Microscopía Confocal , Microscopía Electrónica , Neuroblastoma/patología , Neuroblastoma/ultraestructura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Proteínas tau/genética , Proteínas tau/ultraestructuraRESUMEN
Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.
Asunto(s)
Proteínas de Drosophila/fisiología , Exonucleasas/genética , Longevidad/genética , Proteínas Mitocondriales/fisiología , Ribosomas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Exonucleasas/fisiología , Células Germinativas/metabolismo , Homeostasis , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Células Madre/metabolismoRESUMEN
DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.
Asunto(s)
ADN Primasa/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Enzimas Multifuncionales/metabolismo , Proteína de Replicación A/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Pollos , Cromatina/metabolismo , Cristalografía por Rayos X , ADN Primasa/química , ADN Polimerasa Dirigida por ADN/química , Células HEK293 , Humanos , Modelos Biológicos , Enzimas Multifuncionales/química , Unión Proteica , Dominios Proteicos , Proteína de Replicación A/química , XenopusRESUMEN
PrimPol, (primase-polymerase), the most recently identified eukaryotic polymerase, has roles in both nuclear and mitochondrial DNA maintenance. PrimPol is capable of acting as a DNA polymerase, with the ability to extend primers and also bypass a variety of oxidative and photolesions. In addition, PrimPol also functions as a primase, catalysing the preferential formation of DNA primers in a zinc finger-dependent manner. Although PrimPol's catalytic activities have been uncovered in vitro, we still know little about how and why it is targeted to the mitochondrion and what its key roles are in the maintenance of this multicopy DNA molecule. Unlike nuclear DNA, the mammalian mitochondrial genome is circular and the organelle has many unique proteins essential for its maintenance, presenting a differing environment within which PrimPol must function. Here, we discuss what is currently known about the mechanisms of DNA replication in the mitochondrion, the proteins that carry out these processes and how PrimPol is likely to be involved in assisting this vital cellular process.
Asunto(s)
ADN Primasa/metabolismo , Replicación del ADN , ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Enzimas Multifuncionales/metabolismo , Animales , Núcleo Celular/genética , ADN Primasa/genética , ADN Polimerasa Dirigida por ADN/genética , Humanos , Mitocondrias/genética , Enzimas Multifuncionales/genética , MutaciónRESUMEN
The genetic information in mammalian mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its replication and expression. Many molecules of mitochondrial DNA bear a third strand of DNA, known as "7S DNA," which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for mitochondrial DNA replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with mitochondrial disease, R-loops are of low abundance, and there is mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.
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ADN Mitocondrial/genética , Mitocondrias/genética , Mutación , Ribonucleasa H/genética , Animales , Línea Celular Tumoral , Células Cultivadas , Replicación del ADN , ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Conformación de Ácido Nucleico , Ribonucleasa H/metabolismoRESUMEN
PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.
Asunto(s)
Daño del ADN , ADN Primasa/metabolismo , Replicación del ADN , Nucleósidos/metabolismo , Animales , Biocatálisis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Pollos , Cisplatino/farmacología , Replicación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/metabolismo , Región Variable de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Metilmetanosulfonato/farmacología , Hipermutación Somática de Inmunoglobulina/efectos de los fármacos , Rayos UltravioletaRESUMEN
Translesion synthesis (TLS) employs specialized DNA polymerases to bypass replication fork stalling lesions. PrimPol was recently identified as a TLS primase and polymerase involved in DNA damage tolerance. Here, we identify a novel PrimPol binding partner, PolDIP2, and describe how it regulates PrimPol's enzymatic activities. PolDIP2 stimulates the polymerase activity of PrimPol, enhancing both its capacity to bind DNA and the processivity of the catalytic domain. In addition, PolDIP2 stimulates both the efficiency and error-free bypass of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxoG) lesions by PrimPol. We show that PolDIP2 binds to PrimPol's catalytic domain and identify potential binding sites. Finally, we demonstrate that depletion of PolDIP2 in human cells causes a decrease in replication fork rates, similar to that observed in PrimPol(-/-)cells. However, depletion of PolDIP2 in PrimPol(-/-)cells does not produce a further decrease in replication fork rates. Together, these findings establish that PolDIP2 can regulate the TLS polymerase and primer extension activities of PrimPol, further enhancing our understanding of the roles of PolDIP2 and PrimPol in eukaryotic DNA damage tolerance.
Asunto(s)
Daño del ADN , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Enzimas Multifuncionales/metabolismo , Proteínas Nucleares/metabolismo , Células Cultivadas , ADN/metabolismo , ADN Primasa/antagonistas & inhibidores , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Guanina/análogos & derivados , Humanos , Enzimas Multifuncionales/antagonistas & inhibidores , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments.
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Proteínas Aviares/metabolismo , ADN Primasa/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , G-Cuádruplex , Enzimas Multifuncionales/metabolismo , Animales , Proteínas Aviares/genética , Secuencia de Bases , Línea Celular , Pollos , Ensamble y Desensamble de Cromatina , ADN/química , ADN Primasa/genética , ADN Polimerasa Dirigida por ADN/genética , Epigénesis Genética , Inestabilidad Genómica , Histonas/metabolismo , Datos de Secuencia Molecular , Enzimas Multifuncionales/genética , TransfecciónRESUMEN
PrimPol is a recently identified member of the archaeo-eukaryote primase (AEP) family of primase-polymerases. It has been shown that this mitochondrial and nuclear localized enzyme plays roles in the maintenance of both unperturbed replication fork progression and in the bypass of lesions after DNA damage. Here, we utilized an avian (DT40) knockout cell line to further study the consequences of loss of PrimPol (PrimPol(-/-)) on the downstream maintenance of cells after UV damage. We report that PrimPol(-/-) cells are more sensitive to UV-C irradiation in colony survival assays than Pol η-deficient cells. Although this increased UV sensitivity is not evident in cell viability assays, we show that this discrepancy is due to an enhanced checkpoint arrest after UV-C damage in the absence of PrimPol. PrimPol(-/-) arrested cells become stalled in G2, where they are protected from UV-induced cell death. Despite lacking an enzyme required for the bypass and maintenance of replication fork progression in the presence of UV damage, we show that PrimPol(-/-) cells actually have an advantage in the presence of a Chk1 inhibitor due to their slow progression through S-phase.
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Daño del ADN , ADN Primasa/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Puntos de Control de la Fase G2 del Ciclo Celular , Rayos Ultravioleta , Animales , Muerte Celular , Línea Celular , Proliferación Celular , Supervivencia Celular/efectos de la radiación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Pollos , ADN Primasa/genética , ADN Polimerasa Dirigida por ADN/genética , Fase G2/efectos de la radiación , Técnicas de Inactivación de Genes , Mitosis/efectos de la radiación , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication.
Asunto(s)
ADN Primasa/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Mitocondriales/metabolismo , Enzimas Multifuncionales/metabolismo , Proteína de Replicación A/metabolismo , Cartilla de ADN/biosíntesis , Replicación del ADN , Humanos , Mutagénesis , Antígeno Nuclear de Célula en Proliferación/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína de Replicación A/químicaRESUMEN
PrimPol is a primase-polymerase found in humans, and other eukaryotes, involved in bypassing lesions encountered during DNA replication. PrimPol employs both translesion synthesis and repriming mechanisms to facilitate lesion bypass by the replisome. PrimPol has been reported to be a potential susceptibility gene associated with the development of myopia. Mutation of tyrosine 89 to aspartic acid (PrimPolY89D) has been identified in a number of cases of high myopia, implicating it in the aetiology of this disorder. Here, we examined whether this mutation resulted in any changes in the molecular and cellular activities associated with human PrimPol. We show that PrimPolY89D has a striking decrease in primase and polymerase activities. The hydrophobic ring of tyrosine is important for retaining wild-type extension activity. We also demonstrate that the decreased activity of PrimPolY89D is associated with reduced affinities for DNA and nucleotides, resulting in diminished catalytic efficiency. Although the structure and stability of PrimPolY89D is altered, its fidelity remains unchanged. This mutation also reduces cell viability after DNA damage and significantly slows replication fork rates in vivo. Together, these findings establish that the major DNA replication defect associated with this PrimPol mutant is likely to contribute to the onset of high myopia.
Asunto(s)
ADN Primasa/genética , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Enzimas Multifuncionales/genética , Miopía/genética , Mutación Puntual , ADN/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Enzimas Multifuncionales/química , Enzimas Multifuncionales/metabolismoRESUMEN
PrimPol is a primase-polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol-/- cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.
Asunto(s)
ADN Primasa/química , ADN Polimerasa Dirigida por ADN/química , Enzimas Multifuncionales/química , Animales , Dominio Catalítico , Línea Celular , ADN Primasa/fisiología , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/fisiología , Ensayo de Cambio de Movilidad Electroforética , Humanos , Manganeso/química , Enzimas Multifuncionales/fisiología , Unión Proteica , Proteínas de Xenopus/química , Zinc/químicaRESUMEN
DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.
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Cromosomas Humanos/genética , Aductos de ADN/genética , ADN Primasa/fisiología , Replicación del ADN , ADN Polimerasa Dirigida por ADN/fisiología , Enzimas Multifuncionales/fisiología , Secuencia de Aminoácidos , Animales , Proliferación Celular , Supervivencia Celular , Pollos , Aductos de ADN/química , Aductos de ADN/metabolismo , Daño del ADN , ADN Primasa/química , ADN de Cadena Simple/química , ADN Polimerasa Dirigida por ADN/química , Puntos de Control de la Fase G2 del Ciclo Celular , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Enzimas Multifuncionales/química , Rayos Ultravioleta , XenopusRESUMEN
We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.