Abstract
DNA double-strand breaks (DSBs) are the most severe form of DNA damage, primarily repaired by the non-homologous end joining (NHEJ) pathway. A critical step in this process is DNA synapsis, where the two broken ends are brought together to facilitate timely repair. Deficiencies in NHEJ synapsis can lead to improper DNA end configurations, potentially resulting in chromosomal translocations. NHEJ synapsis is a highly dynamic, multi-protein mediated assembly process. Recent advances in single-molecule techniques have led to significant progress in understanding the molecular mechanisms driving NHEJ synapsis. In this review, we summarize single-molecule methods developed for studying NHEJ synapsis, with a particular focus on the single-molecule fluorescence resonance energy transfer (smFRET) technique. We discuss the various molecular mechanisms of NHEJ synapsis uncovered through these studies and explore the coupling between synapsis and other steps in NHEJ. Additionally, we highlight the strategies, limitations, and future directions for single-molecule studies of NHEJ synapsis.