Repairing DNA double-strand breaks (DSBs) by non-homologous 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 nano-manipulation 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 addition of PAXX extends this lifetime to ~2s. Further addition of XRCC4, XLF and Ligase IV resulted in minute-scale synapsis and led 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 kCal/mol. 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.
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 ∼4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA.
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