Single-molecule techniques in studying the molecular mechanisms of DNA synapsis in non-homologous end-joining repair

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.

Graphical abstract

Keywords

Single-molecule techniques / smFRET / Magnetic tweezers / Non-homologous end joining (NHEJ) / DNA Synapsis

Acknowledgements

The authors would like to thank the members of the Zhao laboratory for helpful discussions and comments on this review. We apologize that many research findings were not directly cited because of the space limitation. This work is supported by the National Natural Science Foundation of China (22104120), the Three Qin Talents Introduction Program for Youths of Shaanxi Province (2023SYJ26), the Fundamental Research Funds for the Central Universities (xtr052023008), and the “Young Talent Support Plan” of Xi’an Jiaotong University to Bailin Zhao; and by the National Natural Science Foundation of China (32400427), the China Postdoctoral Science Foundation (2022M712546), the Fundamental Research Funds for the Central Universities (xzy012023018), and the Postdoctoral Research Project of Shaanxi Province (2023BSHEDZZ07) to Di Liu; and by the Fundamental Research Funds for the Central Universities (xzy022024035) to Chenyang Zhang.

RIGHTS & PERMISSIONS

2025 The Author(s)