RESUMEN
Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.
Asunto(s)
ADN Polimerasa I , Complejo Shelterina , Proteínas de Unión a Telómeros , Telómero , Humanos , Microscopía por Crioelectrón , ADN Polimerasa I/metabolismo , ADN Primasa/metabolismo , ADN Primasa/genética , Modelos Moleculares , Fosforilación , Complejo Shelterina/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.
Asunto(s)
Replicación del ADN , ADN/biosíntesis , Eucariontes/genética , Complejo de Reconocimiento del Origen/metabolismo , Animales , ADN/química , ADN Polimerasa III/química , ADN Polimerasa III/metabolismo , Humanos , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/genética , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a â¼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.
Asunto(s)
ADN Glicosilasas/química , ADN Helicasas/química , ADN Polimerasa Dirigida por ADN/química , ADN/química , Endonucleasas/química , Genoma , Proteínas Hierro-Azufre/química , Animales , Bacterias/genética , Bacterias/metabolismo , ADN/metabolismo , ADN/ultraestructura , Daño del ADN , ADN Glicosilasas/metabolismo , ADN Glicosilasas/ultraestructura , ADN Helicasas/metabolismo , ADN Helicasas/ultraestructura , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/ultraestructura , Espectroscopía de Resonancia por Spin del Electrón , Endonucleasas/metabolismo , Endonucleasas/ultraestructura , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/ultraestructura , Oxidación-Reducción , Unión Proteica , Transducción de Señal , TermodinámicaRESUMEN
Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.
Asunto(s)
ADN Primasa/metabolismo , ADN Primasa/fisiología , Cartilla de ADN/química , Animales , Sitios de Unión , ADN , ADN Primasa/ultraestructura , Cartilla de ADN/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Nucleótidos , Conformación Proteica , Elementos Estructurales de las Proteínas/fisiologíaRESUMEN
This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.
Asunto(s)
ADN Helicasas/genética , ADN Polimerasa II/genética , Replicación del ADN , ADN/genética , Células Eucariotas/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , ADN Helicasas/metabolismo , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa II/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Eucariotas/citología , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismoRESUMEN
During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
Asunto(s)
ADN Primasa , Replicación del ADN , Humanos , ADN Primasa/genética , ADN Primasa/metabolismo , Microscopía por Crioelectrón , ADN Helicasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ADN de Cadena Simple/metabolismoRESUMEN
Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.
Asunto(s)
ADN Primasa/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , AdnB Helicasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Holoenzimas/química , ADN Primasa/genética , ADN Bacteriano , ADN Polimerasa Dirigida por ADN/genética , AdnB Helicasas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Conformación Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Telomere maintenance is essential for the genome integrity of eukaryotes, and this function is underpinned by the two-step telomeric DNA synthesis process: telomere G-overhang extension by telomerase and complementary strand fill-in by DNA polymerase alpha-primase (polα-primase). Compared to the telomerase step, the telomere C-strand fill-in mechanism is less understood. Recent studies have provided new insights into how telomeric single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) and polα-primase coordinate to synthesize the telomeric C-strand for telomere overhang fill-in. Cryogenic electron microscopy (cryo-EM) structures of CST-polα-primase complexes have provided additional insights into how they assemble at telomeric templates and de novo synthesize the telomere C-strand. In this review, we discuss how these latest findings coalesce with existing understanding to develop a human telomere C-strand fill-in mechanism model.
Asunto(s)
ADN Primasa , Telomerasa , Humanos , Telómero , Complejo Shelterina , EucariontesRESUMEN
The replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome's initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins.
Asunto(s)
Reparación del ADN/fisiología , Replicación del ADN/fisiología , ADN/metabolismo , Daño del ADN/fisiología , ADN Primasa/metabolismo , Reparación del ADN/genética , ADN de Cadena Simple/metabolismo , Eucariontes/genética , Células Eucariotas/metabolismo , Origen de Réplica/genética , Origen de Réplica/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
DNA replication commences at eukaryotic replication origins following assembly and activation of bidirectional CMG helicases. Once activated, CMG unwinds the parental DNA duplex and DNA polymerase α-primase initiates synthesis on both template strands. By utilizing an origin-dependent replication system using purified yeast proteins, we have mapped start sites for leading-strand replication. Synthesis is mostly initiated outside the origin sequence. Strikingly, rightward leading strands are primed left of the origin and vice versa. We show that each leading strand is established from a lagging-strand primer synthesized by the replisome on the opposite side of the origin. Preventing elongation of primers synthesized left of the origin blocked rightward leading strands, demonstrating that replisomes are interdependent for leading-strand synthesis establishment. The mechanism we reveal negates the need for dedicated leading-strand priming and necessitates a crucial role for the lagging-strand polymerase Pol δ in connecting the nascent leading strand with the advancing replisome.
RESUMEN
It is now well recognized that the information processing machineries of archaea are far more closely related to those of eukaryotes than to those of their prokaryotic cousins, the bacteria. Extensive studies have been performed on the structure and function of the archaeal DNA replication origins, the proteins that define them, and the macromolecular assemblies that drive DNA unwinding and nascent strand synthesis. The results from various archaeal organisms across the archaeal domain of life show surprising levels of diversity at many levels-ranging from cell cycle organization to chromosome ploidy to replication mode and nature of the replicative polymerases. In the following, we describe recent advances in the field, highlighting conserved features and lineage-specific innovations.
Asunto(s)
Archaea/genética , Proteínas Arqueales/genética , Replicación del ADN , ADN de Archaea/genética , Archaea/fisiología , ADN de Archaea/fisiología , Modelos Moleculares , Unión ProteicaRESUMEN
Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.
Asunto(s)
ADN Polimerasa I , ADN Primasa , Replicación del ADN , Proteínas de Unión a Telómeros , Telómero , Humanos , Telómero/metabolismo , Telómero/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa I/genética , ADN Polimerasa I/química , ADN Primasa/metabolismo , ADN Primasa/genética , ADN Primasa/química , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/genética , Telomerasa/metabolismo , Telomerasa/genéticaRESUMEN
HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.
RESUMEN
Helicase-primase is an interesting target for the therapy of herpes simplex virus (HSV) infections. Since amenamevir is already approved for varicella-zoster virus (VZV) and HSV in Japan and pritelivir has received breakthrough therapy status for the treatment of acyclovir-resistant HSV infections in immunocompromised patients, the target has sparked interest in me-too approaches. Here, we describe the attempt to improve nervous tissue penetration in Phaeno Therapeutics drug candidate HN0037 to target the latent reservoir of HSV by installing less polar moieties, mainly a difluorophenyl instead of a pyridyl group, and replacing the primary sulfonamide with a methyl sulfoximine moiety. However, all obtained stereoisomers exhibited a weaker inhibitory activity on HSV-1 and HSV-2.
Asunto(s)
Antivirales , ADN Primasa , Sulfonamidas , Sulfonamidas/química , Sulfonamidas/farmacología , Sulfonamidas/síntesis química , ADN Primasa/antagonistas & inhibidores , ADN Primasa/metabolismo , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Relación Estructura-Actividad , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/metabolismo , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Humanos , Estructura Molecular , Pruebas de Sensibilidad Microbiana , Relación Dosis-Respuesta a Droga , Iminas/química , Iminas/farmacología , Iminas/síntesis químicaRESUMEN
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.
Asunto(s)
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
BACKGROUND: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). RESULTS: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. CONCLUSIONS: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.
Asunto(s)
Arabidopsis , ADN Primasa , Animales , ADN Primasa/genética , ADN Primasa/química , ADN Primasa/metabolismo , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Arabidopsis/metabolismo , Mitocondrias/metabolismo , Dedos de Zinc , Ribonucleótidos , Replicación del ADN , Bacteriófago T7/genéticaRESUMEN
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.
Asunto(s)
ADN Primasa , ADN Polimerasa Dirigida por ADN , Humanos , ADN Primasa/genética , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Replicación del ADN , ADN/metabolismo , Daño del ADN , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismoRESUMEN
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.
Asunto(s)
Adenocarcinoma del Pulmón , ADN Polimerasa Dirigida por ADN , Humanos , ADN Polimerasa Dirigida por ADN/metabolismo , Células A549 , Cisplatino/farmacología , Peróxido de Hidrógeno/farmacología , Replicación del ADN , Daño del ADN , Adenocarcinoma del Pulmón/genética , ADN Primasa/genética , ADN Primasa/metabolismo , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismoRESUMEN
Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided.
Asunto(s)
Proteínas Bacterianas , Replicación del ADN , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Bacterias/metabolismo , Unión ProteicaRESUMEN
Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication during the S phase. Over the last decade, extensive genome-wide association studies and expression profiling studies of the replicative DNA polymerase genes in human patients have revealed a link between the replicative DNA polymerase genes and various human diseases and disorders including cancer, intellectual disability, microcephalic primordial dwarfism and immunodeficiency. These studies suggest the importance of dissecting the mechanisms involved in the functioning of replicative DNA polymerases in understanding and treating a range of human diseases. Previous studies in Drosophila have established this organism as a useful model to understand a variety of human diseases. Here, we review the studies on Drosophila that explored the link between DNA polymerases and human disease. First, we summarize the recent studies linking replicative DNA polymerases to various human diseases and disorders. We then review studies on replicative DNA polymerases in Drosophila. Finally, we suggest the possible use of Drosophila models to study human diseases and disorders associated with replicative DNA polymerases.