RESUMEN
Ultra-violet (UV) radiation (UVR) causes significant oxidative injury to retinal pigment epithelium (RPE) cells. Obacunone is a highly oxygenated triterpenoid limonoid compound with various pharmacological properties. Its potential effect in RPE cells has not been studied thus far. Here in ARPE-19 cells and primary murine RPE cells, obacunone potently inhibited UVR-induced reactive oxygen species accumulation, mitochondrial depolarization, lipid peroxidation and single strand DNA accumulation. UVR-induced RPE cell death and apoptosis were largely alleviated by obacunone. Obacunone activated Nrf2 signaling cascade in RPE cells, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation. It promoted transcription and expression of antioxidant responsive element-dependent genes. Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout almost reversed obacunone-induced RPE cytoprotection against UVR. Forced activation of Nrf2 cascade, by Keap1 knockout, similarly protected RPE cells from UVR. Importantly, obacunone failed to offer further RPE cytoprotection against UVR in Keap1-knockout cells. In vivo, intravitreal injection of obacunone largely inhibited light-induced retinal damage. Collectively, obacunone protects RPE cells from UVR-induced oxidative injury through activation of Nrf2 signaling cascade.
Asunto(s)
Benzoxepinas/farmacología , Limoninas/farmacología , Degeneración Macular/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Rayos Ultravioleta/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Benzoxepinas/uso terapéutico , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , ADN de Cadena Simple/efectos de los fármacos , ADN de Cadena Simple/efectos de la radiación , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Inyecciones Intravítreas , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Limoninas/uso terapéutico , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/efectos de la radiación , Degeneración Macular/etiología , Degeneración Macular/patología , Ratones , Membranas Mitocondriales/efectos de los fármacos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/genética , Estrés Oxidativo/efectos de la radiación , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/efectos de la radiación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/efectos de la radiaciónRESUMEN
Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.
Asunto(s)
Criptocromos/genética , ADN de Cadena Simple/genética , Desoxirribodipirimidina Fotoliasa/genética , Secuencia de Aminoácidos/genética , Dominio Catalítico/genética , Dominio Catalítico/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , ADN de Cadena Simple/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/efectos de la radiación , Methylobacterium/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/efectos de la radiación , Rhodobacteraceae/genética , Rayos UltravioletaRESUMEN
Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five 'structural' subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD's DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD's affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62's high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ARN/ultraestructura , Factor de Transcripción TFIIH/ultraestructura , Sitios de Unión/efectos de la radiación , Microscopía por Crioelectrón , Daño del ADN/efectos de la radiación , ADN Helicasas/genética , ADN Helicasas/ultraestructura , ADN de Cadena Simple/genética , ADN de Cadena Simple/efectos de la radiación , ADN de Cadena Simple/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , Proteínas de Unión al ARN/genética , Imagen Individual de Molécula , Factor de Transcripción TFIIH/genética , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Proteína de la Xerodermia Pigmentosa del Grupo D/ultraestructuraRESUMEN
The elimination of DNA polymerase eta (pol η) causes discontinuous DNA elongation and fork stalling in UV-irradiated cells. Such alterations in DNA replication are followed by S-phase arrest, DNA double-strand break (DSB) accumulation, and cell death. However, their molecular triggers and the relative timing of these events have not been fully elucidated. Here, we report that DSBs accumulate relatively early after UV irradiation in pol η-depleted cells. Despite the availability of repair pathways, DSBs persist and chromosome instability (CIN) is not detectable. Later on cells with pan-nuclear γH2AX and massive exposure of template single-stranded DNA (ssDNA), which indicate severe replication stress, accumulate and such events are followed by cell death. Reinforcing the causal link between the accumulation of pan-nuclear ssDNA/γH2AX signals and cell death, downregulation of RPA increased both replication stress and the cell death of pol η-deficient cells. Remarkably, DSBs, pan-nuclear ssDNA/γH2AX, S-phase arrest, and cell death are all attenuated by MRE11 nuclease knockdown. Such results suggest that unscheduled MRE11-dependent activities at replicating DNA selectively trigger cell death, but not CIN. Together these results show that pol η-depletion promotes a type of cell death that may be attractive as a therapeutic tool because of the lack of CIN.
Asunto(s)
Roturas del ADN de Doble Cadena/efectos de la radiación , ADN Polimerasa Dirigida por ADN/genética , Histonas/genética , Proteína Homóloga de MRE11/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Muerte Celular/genética , Inestabilidad Cromosómica/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , ADN de Cadena Simple/efectos de la radiación , Humanos , Fase S/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
We reconstituted two biochemical processes that may contribute to UV-induced mutagenesis in vitro and analysed the mutational profiles in the products. One process is translesion synthesis (TLS) by DNA polymerases (Pol) δ, η and ζ, which creates C>T transitions at pyrimidine dimers by incorporating two dAMPs opposite of the dimers. The other process involves spontaneous deamination of cytosine, producing uracil in pyrimidine dimers, followed by monomerization of the dimers by secondary UV irradiation, and DNA synthesis by Pol δ. The mutational spectrum resulting from deamination without translesion synthesis is similar to a mutational signature found in melanomas, suggesting that cytosine deamination encountered by the replicative polymerase has a prominent role in melanoma development. However, CC>TT dinucleotide substitution, which is also commonly observed in melanomas, was produced almost exclusively by TLS. We propose that both TLS-dependent and deamination-dependent mutational processes are likely involved in UV-induced melanoma development.
Asunto(s)
Daño del ADN , ADN de Cadena Simple/efectos de la radiación , Melanoma/genética , Modelos Genéticos , Mutagénesis/efectos de la radiación , Neoplasias Inducidas por Radiación/genética , Dímeros de Pirimidina , Neoplasias Cutáneas/genética , Rayos Ultravioleta/efectos adversos , 5-Metilcitosina/efectos de la radiación , Sistema Libre de Células , Citosina/química , Citosina/efectos de la radiación , Replicación del ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , ADN de Cadena Simple/química , ADN-Citosina Metilasas/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desaminación , Humanos , Melanoma/etiología , Dímeros de Pirimidina/química , Neoplasias Cutáneas/etiología , TranscriptomaRESUMEN
Photoelectrochemical (PEC) sensing techniques have attracted considerable concerns because of the intrinsic merit of complete separation between the excitation light and responsive current but still remain a great challenge for further potential application. It is assigned to the scarcity of photoactive materials with narrow band gap, good biosafety, and high photon-to-electron conversion efficiency and unfavorable processing methods for photoactive materials on indium tin oxide. Herein, we employed a perylene-based polymer (PTC-NH2) with exceptional photoelectrical properties to develop a red-light-driven PEC sensor for ultrasensitive biosensing based on its superior electrostatic intercalation efficiency in double-stranded DNA to that in single-stranded DNA, with DNA adenine methyltransferase (Dam MTase) as the model target. The prepared PTC-NH2 was characterized by Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, and PEC techniques, and the results demonstrated that PTC-NH2 rather than metal oxides/metal sulfides/C3N4/metal complexes enjoyed the prominent capacity of converting light to current. Benefiting from the unique PEC properties of PTC-NH2 and target-initiated hybridization chain reaction (HCR) signal amplification, ultrasensitive detection of Dam MTase was accessibly realized with the detection limit of 0.015 U/mL, which is lower than that of PEC, electrochemical, or fluorescent biosensors previously reported. Furthermore, the proposed PEC sensor has been also applied in screening Dam MTase activity inhibitors. Therefore, the perylene-based PEC sensor exhibits great potential in early accurate diagnosis of DNA methylation-related diseases.
Asunto(s)
Técnicas Biosensibles , ADN/química , Sustancias Intercalantes/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/aislamiento & purificación , ADN de Cadena Simple/efectos de la radiación , Técnicas Electroquímicas/métodos , Humanos , Luz , Perileno/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/química , Compuestos de Estaño/químicaRESUMEN
RATIONALE: Calf-thymus (CT-DNA) is widely used as a binding agent. The commercial samples are known to be "highly polymerized DNA" samples. CT-DNA is known to be fragile in particular upon ultrasonic wave irradiation. Degradation products could have dramatic consequences on its bio-sensing activity, and an accurate determination of the molecular weight distribution and stability of commercial samples is highly demanded. METHODS: We investigated the sensitivity of charge detection mass spectrometry (CDMS), a single-molecule MS method, both with single-pass and ion trap CDMS ("Benner" trap) modes to the determination of the composition and stability (under multiphoton IR irradiation) of calf-thymus DNAs. We also investigated the changes in molecular weight distributions in the course of sonication by irradiating ultrasonic waves to CT-DNA. RESULTS: We report, for the first time, the direct molecular weight (MW) distribution of DNA sodium salt from calf-thymus revealing two populations at high (~10 MDa) and low (~3 MDa) molecular weights. We evidence a transition between the high-MW to the low-MW distribution, confirming that the low-MW distribution results from degradation of CT-DNA. Finally, we report also IRMPD experiments carried out on trapped single-stranded linear DNAs from calf-thymus allowing extraction of their activation energy for unimolecular dissociation. CONCLUSIONS: We show that single-pass CDMS is a direct, efficient and accurate MS-based approach to determine the composition of calf-thymus DNAs. Furthermore, ion trap CDMS allows us to evaluate the stability (both under multiphoton IR irradiation and in the course of sonication by irradiating ultrasonic wave) of calf-thymus DNAs.
Asunto(s)
ADN/análisis , ADN/química , Espectrometría de Masas/métodos , ADN/efectos de la radiación , ADN de Cadena Simple/análisis , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de la radiación , Rayos Infrarrojos , Peso Molecular , SonicaciónRESUMEN
Herein, by directly using Watson-Crick base pairing, a highly ordered and field-free three-dimensional (3D) DNA nanostructure is self-assembled by azobenzene (azo)-functionalized DNA nippers in a few minutes, which was applied as a 3D DNA nanomachine with an improved movement efficiency compared to traditional Au-based 3D nanomachines due to the organized and high local concentration of nippers on homogeneous DNA nanostructure. Once microRNA (miRNA) interacts with the 3D nanomachine, the nippers "open" to hybridize with the miRNA. Impressively, photoisomerization of the azo group induces dehybridization/hybridization of the nippers and miRNA under irradiation at different wavelengths, which easily solves one main technical challenge of DNA nanotechnology and biosensing: reversible locomotion in one step within 10 min. As a proof of concept, the described 3D machine is successfully applied in the rapid single-step detection of a biomarker, which gives impetus to the design of new generations of mechanical devices beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.
Asunto(s)
ADN de Cadena Simple/química , Nanoestructuras/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , 2,2'-Dipiridil/efectos de la radiación , Emparejamiento Base , Biomarcadores/análisis , Técnicas Biosensibles/métodos , Línea Celular Tumoral , ADN de Cadena Simple/genética , ADN de Cadena Simple/efectos de la radiación , Colorantes Fluorescentes/química , Colorantes Fluorescentes/efectos de la radiación , Humanos , MicroARNs/análisis , Movimiento , Nanoestructuras/efectos de la radiación , Nanotecnología/métodos , Hibridación de Ácido Nucleico , Compuestos Organometálicos/química , Compuestos Organometálicos/efectos de la radiaciónRESUMEN
Two-photon photoluminescence (TPPL) emission spectra of DNA-gold nanoparticle (AuNP) monoconjugates and the corresponding DNA-linked AuNP dimers are obtained by photon time-of-flight spectroscopy. This technique is combined with two-photon photoluminescence fluctuation correlation spectroscopy (TPPL-FCS) to simultaneously monitor the optical and hydrodynamic behaviour of these nano-assemblies in solution, with single-particle sensitivity and microsecond temporal resolution. In this study, the AuNPs have an average core diameter of 12â nm, which renders their dark-field plasmonic light scattering too weak for single-particle imaging. Moreover, as a result of the lack of plasmonic coupling in the dimers, the optical extinction, scattering and photoluminescence spectra of the DNA-AuNP complexes are not sufficiently different to distinguish between monomers and dimers. The use of TPPL-FCS successfully addresses these bottlenecks and enables the distinction between AuNP monomers and AuNP dimers in solution by measurement of their hydrodynamic rotational and translational diffusion.
Asunto(s)
ADN de Cadena Simple/química , Oro/química , Hidrodinámica , Nanopartículas del Metal/química , ADN de Cadena Simple/efectos de la radiación , Difusión , Luminiscencia , Nanopartículas del Metal/efectos de la radiación , Tamaño de la Partícula , Fotones , Dispersión de Radiación , Espectrofotometría/métodosRESUMEN
In this work, direct DNA damage induced by low-energy electrons (sub-keV) is simulated using a Monte Carlo method. The characteristics of the present simulation are to consider the new mechanism of DNA damage due to dissociative electron attachment (DEA) and to allow determining damage to specific bases (i.e., adenine, thymine, guanine, or cytosine). The electron track structure in liquid water is generated, based on the dielectric response model for describing electron inelastic scattering and on a free-parameter theoretical model and the NIST database for calculating electron elastic scattering. Ionization cross sections of DNA bases are used to generate base radicals, and available DEA cross sections of DNA components are applied for determining DNA-strand breaks and base damage induced by sub-ionization electrons. The electron elastic scattering from DNA components is simulated using cross sections from different theoretical calculations. The resulting yields of various strand breaks and base damage in cellular environment are given. Especially, the contributions of sub-ionization electrons to various strand breaks and base damage are quantitatively presented, and the correlation between complex clustered DNA damage and the corresponding damaged bases is explored. This work shows that the contribution of sub-ionization electrons to strand breaks is substantial, up to about 40-70%, and this contribution is mainly focused on single-strand break. In addition, the base damage induced by sub-ionization electrons contributes to about 20-40% of the total base damage, and there is an evident correlation between single-strand break and damaged base pair A-T.
Asunto(s)
Daño del ADN , Electrones/efectos adversos , Método de Montecarlo , ADN de Cadena Simple/efectos de la radiaciónRESUMEN
The nucleotide excision repair system removes a wide variety of DNA lesions from the human genome, including photoproducts induced by ultraviolet (UV) wavelengths of sunlight. A defining feature of nucleotide excision repair is its dual incision mechanism, in which two nucleolytic incision events on the damaged strand of DNA at sites bracketing the lesion generate a damage-containing DNA oligonucleotide and a single-stranded DNA gap approximately 30 nucleotides in length. Although the early events of nucleotide excision repair, which include lesion recognition and the dual incisions, have been explored in detail and are reasonably well understood, the fate of the single-stranded DNA gaps and excised oligonucleotide products of repair have not been as extensively examined. In this review, recent findings that address these less-explored aspects of nucleotide excision repair are discussed and support the concept that postincision gap and excised oligonucleotide processing are critical steps in the cellular response to DNA damage induced by UV light and other environmental carcinogens. Defects in these latter stages of repair lead to cell death and other DNA damage signaling responses and may therefore contribute to a number of human disease states associated with exposure to UV wavelengths of sunlight, including skin cancer, aging and autoimmunity.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN de Cadena Simple/efectos de la radiación , Rayos Ultravioleta , Muerte Celular/efectos de la radiación , HumanosRESUMEN
DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.
Asunto(s)
ADN Helicasas/genética , Inestabilidad Genómica/genética , Linfoma de Células T/genética , Animales , Apoptosis/genética , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , ADN de Cadena Simple/efectos de la radiación , Modelos Animales de Enfermedad , Rayos gamma , Inestabilidad Genómica/efectos de la radiación , Células Madre Hematopoyéticas/patología , Humanos , Linfocitos/patología , Linfocitos/efectos de la radiación , Linfoma de Células T/patología , Ratones , MutaciónRESUMEN
Nucleotide excision repair and the ATR-mediated DNA damage checkpoint are two critical cellular responses to the genotoxic stress induced by ultraviolet (UV) light and are important for cancer prevention. In vivo genetic data indicate that these global responses are coupled. Aziz Sancar et al. developed an in vitro coupled repair-checkpoint system to analyze the basic steps of these DNA damage stress responses in a biochemically defined system. The minimum set of factors essential for repair-checkpoint coupling include damaged DNA, the excision repair factors (XPA, XPC, XPF-ERCC1, XPG, TFIIH, RPA), the 5'-3' exonuclease EXO1, and the damage checkpoint proteins ATR-ATRIP and TopBP1. This coupled repair-checkpoint system was used to demonstrate that the ~30 nucleotide single-stranded DNA (ssDNA) gap generated by nucleotide excision repair is enlarged by EXO1 and bound by RPA to generate the signal that activates ATR.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN de Cadena Simple/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Rayos Ultravioleta , HumanosRESUMEN
Experimental studies showed that high energy radiation induced base release and DNA backbone breaks mainly occur at the neighboring 5' nucleotide when a single-stranded DNA is modified by radiosensitizing 5-halogenated deoxyuridines. However, no mechanism can be used to interpret these experimental observations. To better understand the radiosensitivity of 5-halogenated deoxyuridines, mechanisms involving hydrogen abstraction by the uracil-5-yl radical from the C2' and C3' positions of an adjacent nucleotide separately followed by the C3'-O3' or N-glycosidic bond rupture and the P-O3' bond breakage are investigated in the DNA sequence 5'-TU(â¢)-3' employing density functional theory calculations in the present study. It is found that hydrogen abstractions from both positions are comparable with the one from the C2' site slightly more favorable. The N-glycosidic bond cleavage in the neighboring 5' nucleotide following the internucleotide C2'-Ha abstraction is estimated to have the lowest activation free energies, indicating that the adjacent 5' base release dominates electron induced damage to single-stranded DNA incorporated by 5-halogenated deoxyuridines. Relative to the P-O3' bond breakage after the internucleotide C3'-H abstraction, the C3'-O3' bond rupture in the neighboring 5' nucleotide following the internucleotide C2'-Ha abstraction is predicted to have a lower activation free energy, implying that single-stranded DNA backbone breaks are prone to occur at the C3'-O3' bond site. The 5'-TU(â¢)-3' species has substantial electron affinity and can even capture a hydrated electron, forming the 5'-TU(-)-3' anion. However, the electron induced C3'-O3' bond rupture in 5'-TU(-)-3' anion via a pathway of internucleotide proton abstraction is only minor in both the gas phase and aqueous solution. The present theoretical predictions can interpret rationally experimental observations, thereby demonstrating that the mechanisms proposed here are responsible for high energy radiation induced damage to single-stranded DNA incorporated by radiosensitizing 5-halogenated deoxyuridines. By comparing with previous results, our work proves that the radiosensitizing action of 5-bromo-2-deoxyuridine is not weaker but stronger than its isomer 6-bromo-2-deoxyuridine on the basis of the available data.
Asunto(s)
Daño del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de la radiación , Desoxiuridina/química , Desoxiuridina/efectos de la radiación , Teoría Cuántica , Humanos , Cinética , TermodinámicaRESUMEN
Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.
Asunto(s)
ADN de Cadena Simple/genética , ADN Polimerasa Dirigida por ADN/genética , Microondas , Técnicas de Amplificación de Ácido Nucleico , ADN Circular/genética , ADN Circular/efectos de la radiación , ADN de Cadena Simple/efectos de la radiación , ADN Polimerasa Dirigida por ADN/efectos de la radiación , ARN/efectos de la radiaciónRESUMEN
The photoexcitation of DNA strands triggers extremely complex photoinduced processes, which cannot be understood solely on the basis of the behavior of the nucleobase building blocks. Decisive factors in DNA oligomers and polymers include collective electronic effects, excitonic coupling, hydrogen-bonding interactions, local steric hindrance, charge transfer, and environmental and solvent effects. This chapter surveys recent theoretical and computational efforts to model real-world excited-state DNA strands using a variety of established and emerging theoretical methods. One central issue is the role of localized vs delocalized excitations and the extent to which they determine the nature and the temporal evolution of the initial photoexcitation in DNA strands.
Asunto(s)
ADN/efectos de la radiación , Modelos Teóricos , Procesos Fotoquímicos , Adenina/química , Adenina/efectos de la radiación , Emparejamiento Base/efectos de la radiación , Citosina/química , Citosina/efectos de la radiación , ADN/química , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de la radiación , Electrones , Transferencia de Energía , Guanina/química , Guanina/efectos de la radiación , Enlace de Hidrógeno , Conformación de Ácido Nucleico , Timina/química , Timina/efectos de la radiación , Uracilo/química , Uracilo/efectos de la radiaciónRESUMEN
Ultrafast laser experiments on carefully selected DNA model compounds probe the effects of base stacking, base pairing, and structural disorder on excited electronic states formed by UV absorption in single and double DNA strands. Direct π-orbital overlap between two stacked bases in a dinucleotide or in a longer single strand creates new excited states that decay orders of magnitude more slowly than the generally subpicosecond excited states of monomeric bases. Half or more of all excited states in single strands decay in this manner. Ultrafast mid-IR transient absorption experiments reveal that the long-lived excited states in a number of model compounds are charge transfer states formed by interbase electron transfer, which subsequently decay by charge recombination. The lifetimes of the charge transfer states are surprisingly independent of how the stacked bases are oriented, but disruption of π-stacking, either by elevating temperature or by adding a denaturing co-solvent, completely eliminates this decay channel. Time-resolved emission measurements support the conclusion that these states are populated very rapidly from initial excitons. These experiments also reveal the existence of populations of emissive excited states that decay on the nanosecond time scale. The quantum yield of these states is very small for UVB/UVC excitation, but increases at UVA wavelengths. In double strands, hydrogen bonding between bases perturbs, but does not quench, the long-lived excited states. Kinetic isotope effects on the excited-state dynamics suggest that intrastrand electron transfer may couple to interstrand proton transfer. By revealing how structure and non-covalent interactions affect excited-state dynamics, on-going experimental and theoretical studies of excited states in DNA strands can advance understanding of fundamental photophysics in other nanoscale systems.
Asunto(s)
Adenina/efectos de la radiación , Citosina/efectos de la radiación , ADN/efectos de la radiación , Guanina/efectos de la radiación , Timina/efectos de la radiación , Uracilo/efectos de la radiación , Adenina/química , Emparejamiento Base/efectos de la radiación , Citosina/química , ADN/química , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de la radiación , Transferencia de Energía , Guanina/química , Rayos Láser , Conformación de Ácido Nucleico , Procesos Fotoquímicos , Protones , Análisis Espectral , Timina/química , Factores de Tiempo , Rayos Ultravioleta , Uracilo/químicaRESUMEN
Absorption of UV light by nucleic acids can lead to damaging photoreactions, which may ultimately lead to mutations of the genetic code. The complexity of the photodynamical behavior of nucleobases in the DNA double-helix provides a great challenge to both experimental and computational chemists studying these processes. Starting from the initially excited states, the main question regards the understanding of the subsequent relaxation processes, which can either utilize monomer-like deactivation pathways or lead to excitonic or charge transfer species with new relaxation dynamics. After a review of photophysical processes in single nucleobases we outline the theoretical background relevant for interacting chromophores and assess a large variety of computational approaches relevant for the understanding of the nature and dynamics of excited states of DNA. The discussion continues with the analysis of calculations on excitonic and charge transfer states followed by the presentation of the dynamics of excited-state processes in DNA. The review is concluded by topics on proton transfer in DNA and photochemical dimer formation of nucleobases.
Asunto(s)
ADN/química , ADN/efectos de la radiación , Electrones , Rayos Ultravioleta , Absorción de Radiación , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de la radiación , Transporte de Electrón , Modelos Moleculares , Procesos Fotoquímicos , FotonesRESUMEN
The porphyrin-based photosensitizers capable of binding to DNA are perspective drug candidates. Here we report the interactions with calf thymus DNA of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (P1) and its derivatives containing Zn(II) or Ni(II) in the coordination sphere. These interactions were studied with absorption and circular dichroism spectroscopy. NiP1 and ZnP1 formed different types of complexes with DNA. NiP1 intercalated into the double helix, whereas ZnP1 bound the DNA groove. Compound P1 displayed both binding modes. The ZnP1-DNA binding constant was approximately three times smaller than the respective values for P1-DNA and NiP1-DNA complexes. Light induced degradation of the reactive oxygen species (ROS) trap 1,3-diphenylisobenzofuran in the presence of P1 and its metal derivatives revealed that NiP1 was a weaker photooxidative agent, whereas P1 and ZnP1 generated ROS to similar extents. Nevertheless, the DNA photodamaging effect of ZnP1 was the most pronounced. Illumination of the supercoiled plasmid caused single-strand DNA photocleavage in the presence of P1 and ZnP1; double strand breaks were detectable with micromolar concentrations of ZnP1. The concentration of ZnP1 required for plasmid photonicking was two times smaller than that of P1 and ~20 times lower than that for NiP1. Thus, the modes of P1, NiP1 and ZnP1 binding to DNA determine the differential photodamaging potency of these porphyrins. A greater accessibility to the solvent of the groove binder ZnP1, compared to the shielded intercalator NiP1 and the intercalated P1 molecules, allows for an efficient local generation of ROS followed by DNA photocleavage.
Asunto(s)
ADN de Cadena Simple/química , Metaloporfirinas/química , Níquel/química , Fármacos Fotosensibilizantes/química , Compuestos de Piridinio/química , Zinc/química , Secuencia de Aminoácidos , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/efectos de la radiación , Metaloporfirinas/síntesis química , Metaloporfirinas/farmacología , Metaloporfirinas/efectos de la radiación , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/efectos de la radiación , Compuestos de Piridinio/síntesis química , Rayos UltravioletaRESUMEN
The yeast Exo1p nuclease functions in multiple cellular roles: resection of DNA ends generated during recombination, telomere stability, DNA mismatch repair, and expansion of gaps formed during the repair of UV-induced DNA damage. In this study, we performed high-resolution mapping of spontaneous and UV-induced recombination events between homologs in exo1 strains, comparing the results with spontaneous and UV-induced recombination events in wild-type strains. One important comparison was the lengths of gene conversion tracts. Gene conversion events are usually interpreted as reflecting heteroduplex formation between interacting DNA molecules, followed by repair of mismatches within the heteroduplex. In most models of recombination, the length of the gene conversion tract is a function of the length of single-stranded DNA generated by end resection. Since the Exo1p has an important role in end resection, a reduction in the lengths of gene conversion tracts in exo1 strains was expected. In accordance with this expectation, gene conversion tract lengths associated with spontaneous crossovers in exo1 strains were reduced about twofold relative to wild type. For UV-induced events, conversion tract lengths associated with crossovers were also shorter for the exo1 strain than for the wild-type strain (3.2 and 7.6 kb, respectively). Unexpectedly, however, the lengths of conversion tracts that were unassociated with crossovers were longer in the exo1 strain than in the wild-type strain (6.2 and 4.8 kb, respectively). Alternative models of recombination in which the lengths of conversion tracts are determined by break-induced replication or oversynthesis during strand invasion are proposed to account for these observations.