Single-Molecule Observations of DNA Double-Strand Break Repair by the Human Nonhomologous End-Joining Pathway

In human cells, DNA double-strand breaks (DSBs) are primarily repaired via the nonhomologous end-joining (NHEJ) pathway, which involves the recruitment of various proteins that stimulate the synapsis and ligation of broken chromosomes. NHEJ is a core component of adaptive immunity, and dysfunctional NHEJ has been linked to premature aging, cancer, and neurodegeneration. Given the clinical importance of this system, it is surprising that the exact mechanism by which NHEJ pairs together DNA strands and promotes appropriate rejoining remains poorly defined. Here we employed advanced single-molecule biophysical approaches to address the mechanism of DSB repair by the NHEJ pathway. We used in vivo and in vitro super-resolution microscopy to define the organization and interactions of NHEJ repair proteins at DSB sites. We observed that the core NHEJ ligation complex forms filaments adjacent to DSBs, suggesting that these filaments increase cross sectional area to pair breaks in space. Additionally, we showed using single-molecule fluorescence resonance energy transfer (smFRET) that the filaments formed by the core NHEJ proteins act to join together DNA ends, and that the interaction is highly dynamic. Furthermore, when DSBs are generated, they are known to leave chemical adducts and damages bases on DNA ends, leading to deletions in the error prone process of NHEJ. We believe the core NHEJ ligation complex to have inherently high fidelity, and use a smFRET assay to establish its ability to detect lesions on the DNA ends, as well as sense mismatched bases in short overhangs. We also find that longer ends with less homology pair poorly, and trigger dissociation of synapsed DNA ends, suggesting how error is introduced in vivo if repair is not completed quickly. Collectively, my work defines how NHEJ is able to pair together broken chromosomes with filaments nucleated form the core NHEJ ligation complex, and how fidelity is improved through the detection of errors in end chemistry and base mismatches in overhangs.