RESUMEN
The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.
Asunto(s)
ADN , Timina ADN Glicosilasa , Citosina/química , Citosina/metabolismo , ADN/análisis , ADN/genética , ADN/metabolismo , Reparación del ADN , Humanos , Oligonucleótidos , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Timina/metabolismo , Timina ADN Glicosilasa/análisis , Timina ADN Glicosilasa/genética , Timina ADN Glicosilasa/metabolismo , Uracilo/químicaRESUMEN
Effective detection of thymine DNA glycosylase (TDG) activity is extremely crucial and urgent for epigenetic research. Herein, a novel label-free electrogenerated chemiluminescence (ECL) biosensing method was developed for the detection of TDG activity using DNA-functionalized gold nanoparticles (DNA-AuNPs) triggered hybridization chain reaction (HCR). In this assay, the thiol modified hairpin probe DNA (hp-DNA) with 5' overhangs and one mismatched base pair of guanines: thymine (G: T) in the stem part was boned onto gold electrode. TDG specifically removed T base of the G: T mismatch to produce apyrimidinic (AP) sites through the N-glycosidic bond hydrolysis. The AP site was then cleaved by the catalysis of Endonuclease IV (EnIV) to generate dsDNA containing a free 3' end in the long sequence, which serves as a complementary sequence to hybridize with the specific sequence (ssDNA1) of DNA-AuNPs. Then, the functionalized DNA-AuNPs with initiator strands (ssDNA2) could trigger HCR to form nicked double helices DNA polymer which can embed numerous ECL indicator, Ru(phen)32+, resulting in significantly increased ECL signal. The proposed strategy combined the amplification function of DNA-AuNPs triggered HCR and the inherent high sensitivity of the ECL technique, a detection limit of 1.1â¯×â¯10-5 U/µL (0.0028â¯ng/mL) for TDG determination was obtained. In addition, this method was successfully applied to evaluate TDG activity in cancer cell, which provides great possibility for TDG activity assay in related clinical diagnostics.
Asunto(s)
Técnicas Biosensibles , Neoplasias de la Mama/metabolismo , ADN/química , Oro/química , Mediciones Luminiscentes , Nanopartículas del Metal/química , Timina ADN Glicosilasa/análisis , Neoplasias de la Mama/patología , Técnicas Electroquímicas , Femenino , Humanos , Timina ADN Glicosilasa/metabolismoRESUMEN
Thymine DNA glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation, which also plays an essential role in DNA demethylation. In this work, the novel iridium(III) complex 1 with an anchor tail was synthesized and employed to construct a G-quadruplex-based assay for detecting TDG activity in aqueous solution by using the mismatched base excising property of TDG with T4 DNA ligase and phi29 DNA polymerase, in concert with the rolling circle amplification (RCA) strategy. The assay achieved a detection limit of 0.048UmL(-)(1) (0.012ngmL(-1)), and showed high selectivity towards TDG even in the presence of other proteins and enzymes. Additionally, the assay could function in diluted cellular debris.
Asunto(s)
Sondas de ADN/genética , G-Cuádruplex , Mediciones Luminiscentes/instrumentación , Técnicas de Sonda Molecular/instrumentación , Timina ADN Glicosilasa/análisis , Timina ADN Glicosilasa/genética , Diseño de Equipo , Análisis de Falla de Equipo , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A triple-signal amplification strategy was proposed for highly sensitive and selective detection of thymine DNA glycosylase (TDG) by coupling a dendrimer-like DNA label with the electrochemical method and quantum dots (QDs) tagging. The DNA-QDs dendrimer-like superstructure was designed by DNA hybridization and covalent assembling. Benefiting from outstanding performance of the amplification strategy, this assay showed high sensitivity, extraordinary stability, and easy operation. The limit of detection could reach 0.00003 U µL(-1) with a splendid specificity. The TDG content in different concentration of HeLa cell was also determined. This assay opens a new horizon for both qualitative and quantitative detection of TDG, holding great promise for potential application in cancer cell research and clinical diagnostics.
Asunto(s)
Técnicas Biosensibles/métodos , ADN/química , Dendrímeros/química , Pruebas de Enzimas/métodos , Puntos Cuánticos/química , Timina ADN Glicosilasa/metabolismo , ADN/metabolismo , Técnicas Electroquímicas/métodos , Células HeLa , Humanos , Límite de Detección , Modelos Moleculares , Reproducibilidad de los Resultados , Timina ADN Glicosilasa/análisisRESUMEN
A novel homogeneous fluorescence assay strategy for highly sensitive detection of thymine DNA glycosylase (TDG) enzyme activity based on the exonuclease-mediated signal amplification reaction was reported.
Asunto(s)
Exonucleasas/metabolismo , Espectrometría de Fluorescencia , Timina ADN Glicosilasa/análisis , Línea Celular , Sondas de ADN/metabolismo , Células HeLa , Humanos , Células MCF-7 , Técnicas de Amplificación de Ácido Nucleico , Timina ADN Glicosilasa/genética , Timina ADN Glicosilasa/metabolismo , Temperatura de TransiciónRESUMEN
1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.
Asunto(s)
Pollos/genética , ADN/química , Análisis para Determinación del Sexo/métodos , Timina ADN Glicosilasa/análisis , Animales , Proteínas Aviares/genética , Proteínas de Unión al ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Marcadores Genéticos , Genoma , Reacción en Cadena de la PolimerasaRESUMEN
Cytosine methylation is a vital biology event. However, it is also the source of genomic instability due to deamination of 5'-methylcytosine by spontaneous hydrolysis, which produces thymine and results in G:T mismatches. Thymine DNA glycosylase and methyl-CpG-binding protein 4 are major DNA glycosylases involved in the mismatch repair progress, and their activities have been measured in many related researches. In this study, we developed a convenient spectrometric assay system for specific and quantitative measurement of intracellular DNA glycosylase activity. A G:T mismatch was introduced into the upstream region of firefly luciferase-coding sequence in the pGL3-control plasmid. Only if the G:T mismatches were repaired to G:C, will luciferase be expressed in transfected cells. By measuring luciferase activity, which is simple and convenient, the intracellular DNA glycosylase activity can be determined.
Asunto(s)
Endodesoxirribonucleasas/análisis , Espectrometría de Fluorescencia/métodos , Timina ADN Glicosilasa/análisis , Animales , Disparidad de Par Base , Línea Celular Tumoral , Reparación del ADN , Genes Reporteros , Vectores Genéticos , Luciferasas de Luciérnaga/metabolismo , Luciferasas de Renilla/análisis , Ratones , Plásmidos , Timina/metabolismoRESUMEN
Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9-Rad1-Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67-110 of hTDG, and Val74 of hTDG plays an important role in the TDG-Hus1 interaction. In contrast to the core domain of hTDG (residues 110-308), hTDG(67-308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER.