RESUMEN
DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.
Asunto(s)
Reparación de la Incompatibilidad de ADN/genética , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas MutL/metabolismo , Disparidad de Par Base/genética , Proteína 9 Asociada a CRISPR/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Endodesoxirribonucleasas/metabolismo , Inestabilidad Genómica/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismoRESUMEN
The combination of single-molecule fluorescence and nanomanipulation techniques into a single experimental platform enables one to carry out correlative analysis of the composition and the activity of complex, multicomponent molecular systems. Here we describe implementation and calibration of such a combined system allowing simultaneous single-molecule force spectroscopy and fluorescence imaging of proteins acting on the DNA using magnetic trapping coupled with fluorescence excitation based on a Total Internal Reflection (TIR), or evanescent, field. We propose a simple and robust in situ method for calibration of the TIR field depth against the mechanical properties of nanomanipulated DNA, and which is made possible by the fact that the magnetic bead used to trap and nanomanipulate DNA and measure its conformation also exhibits autofluorescence in the TIR field. Indeed, the fact that the bead size is on the 1-micron scale does not preclude sensitive probing of an intensity field which decays exponentially on the 0.1micron-scale. We demonstrate the usefulness of this approach by mapping out TIR field depth as a function of the angle of incidence of the illuminating laser at the glass-water interface and showing that one recovers the expected theoretical relationship between field depth and angle of incidence.
Asunto(s)
ADN/química , Nanotecnología/métodos , Imagen Individual de Molécula/métodos , Espectrometría de Fluorescencia/métodos , CalibraciónRESUMEN
Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Técnicas Genéticas/instrumentación , Animales , Proteínas de Unión al Calcio/metabolismo , Emparejamiento Cromosómico , ADN/genética , ADN/metabolismo , ADN Ligasa (ATP)/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas de Restricción del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Fosforilación , Células Sf9 , SpodopteraRESUMEN
We characterize in real time the composition and catalytic state of the initial Escherichia coli transcription-coupled repair (TCR) machinery by using correlative single-molecule methods. TCR initiates when RNA polymerase (RNAP) stalled by a lesion is displaced by the Mfd DNA translocase, thus giving repair components access to the damage. We previously used DNA nanomanipulation to obtain a nanomechanical readout of protein-DNA interactions during TCR initiation. Here we correlate this signal with simultaneous single-molecule fluorescence imaging of labeled components (RNAP, Mfd or RNA) to monitor the composition and localization of the complex. Displacement of stalled RNAP by Mfd results in loss of nascent RNA but not of RNAP, which remains associated with Mfd as a long-lived complex on the DNA. This complex translocates at â¼4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA.