RESUMEN
Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.
Asunto(s)
Replicación del ADN , ADN Mitocondrial/metabolismo , Eliminación de Secuencia , Imagen Individual de Molécula/métodos , Envejecimiento/genética , ADN Helicasas/genética , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
We report a method for the sensitive measurement of genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-ß-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 µL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 µL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 µL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).
Asunto(s)
ADN Bacteriano/análisis , Genoma Bacteriano , Nanotecnología/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Genoma Bacteriano/genética , HumanosRESUMEN
Genome sequencing studies have revealed a number of cancer-associated mutations in the telomere-binding factor POT1. Here, we show that when combined with p53 deficiency, depletion of murine POT1a in common lymphoid progenitor cells fosters genetic instability, accelerates the onset, and increases the severity of T cell lymphomas. In parallel, we examined human and mouse cells carrying POT1 mutations found in cutaneous T cell lymphoma (CTCL) patients. Inhibition of POT1 activates ATR-dependent DNA damage signaling and induces telomere fragility, replication fork stalling, and telomere elongation. Our data suggest that these phenotypes are linked to impaired CST (CTC1-STN1-TEN1) function at telomeres. Lastly, we show that proliferation of cancer cells lacking POT1 is enabled by the attenuation of the ATR kinase pathway. These results uncover a role for defective telomere replication during tumorigenesis.
Asunto(s)
Carcinogénesis/metabolismo , Carcinogénesis/patología , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Estrés Fisiológico , Telómero/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad , Inestabilidad Genómica , Células Progenitoras Linfoides/metabolismo , Linfoma Cutáneo de Células T/genética , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/patología , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Complejo Shelterina , Proteínas de Unión a Telómeros , Timo/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In this paper we present the development of an optical tweezers platform capable of creating on-demand dynamic microbead arrays for the multiplexed detection of biomolecules. We demonstrate the use of time-shared optical tweezers to dynamically assemble arrays of sensing microspheres, while simultaneously recording fluorescence signals in real time. The detection system is able to achieve multiplexing by using quantum dot nanocrystals as both signaling probes and encoding labels on the surface of the trapped microbeads. The encoding can be further extended by using a range of bead sizes. Finally, the platform is used to detect and identify three genes expressed by pathogenic strains of Escherichia coli O157:H7. The in situ actuation enabled by the optical tweezers, combined with multiplexed fluorescence detection offers a new tool, readily adaptable to biosensing applications in microfluidic devices, and could potentially enable the development of on-demand diagnostics platforms.