Live cell single-molecule imaging to study DNA repair in human cells

Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells

“…To analyze the recruitment of the tagged NHEJ factors to DNA lesions in real time, we performed laser micro-irradiation (LMI) experiments using a 405 nm laser of cells sensitized with Hoechst dye. While LMI induces complex DNA lesions, including DSBs, it provides consistent experimental conditions (pulse length, laser power) and allows analysis of NHEJ factor localization to site of DNA damage with minimal time delay after induction of DNA damage 20,25 . All tagged NHEJ factors were robustly recruited to LMI-induced damage sites (Fig.…”

“…To analyze the diffusion dynamics of individual HaloTagged NHEJ factors, we carried out highly inclined laminated optical sheet (HILO) microscopy of cells sparsely labeled with a fluorescent HaloTag ligand in combination with single-particle tracking (SPT, Movie S5-S9) 20, 32,33 . Under these conditions only a small fraction of the total cellular proteins was labeled, ranging from 0.01% for Halo-Ku70 to 0.6% for Halo-XLF (Fig.…”

“…Because NHEJ factors are both highly abundant and only small numbers (two to four) of the core NHEJ proteins are recruited to DNA breaks, it has been challenging to detect their recruitment to DSBs in living cells 20 . In addition, well established methods to analyze the recruitment of DNA repair factors to sites of DNA damage in real time, for example laser microirradiation, generate complex DNA lesions, making it difficult to interpret experimental outcomes [23][24][25] .…”

“…The key assumption is that the immobilization of individual NHEJ factors in response to treatment with DNA damaging agents is caused by their binding to DSBs. The single-molecule approach has two key advantages: individual molecules can be detected making it possible to visualize the recruitment of factors that are not present at breaks in large numbers, and small molecule drugs can be used to induce a titratable number of chemically defined DNA lesions (as opposed to the complex lesions that result from LMI) 20, 23–25 ,. Using this approach, we analyze the temporal and compositional maturation of NHEJ factors bound to DNA breaks and defined of the overall kinetics and repair capacity of the NHEJ pathway in living cells.…”

“…A key challenge has been the lack of tools to study the binding of NHEJ factors to chemically well-defined breaks in living cells. Live cell imaging of laser micro-irradiation (LMI) induced DNA damage has been a useful approach to visualize the recruitment of DNA repair factors to spatially and temporally well controlled DNA lesions in real time [20][21][22] . However, LMI induces complex DNA lesions that include DSBs, single-strand breaks, and base damage and the relative contribution of the various types of DNA damage to the recruitment of the repair factors is impossible to disentangle [23][24][25] .…”