Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4

DNA double-strand breaks are among the most severe forms of DNA damage. Nonhomologous end joining (NHEJ) is a primary repair pathway in eukaryotic cells. While biochemical studies have identified key proteins involved in NHEJ, the process remains poorly understood in living cells. Most prior work has focused on isolated proteins or in vitro systems. This limits the understanding of how these proteins function together in real-time within a cellular environment. The lack of tools to visualize NHEJ proteins at DNA damage sites has hindered progress. Recent advances in laser-based techniques have allowed for the induction of DNA breaks without visible nuclear damage. This has opened new avenues to study DNA repair kinetics in vivo. However, the dynamic behavior of NHEJ proteins at DNA ends remains unclear. This gap motivated researchers to investigate how NHEJ complexes assemble and disassemble in living cells.

The goal of this work was to examine the behavior of NHEJ proteins in living cells during DNA repair. The researchers aimed to determine whether DNA repair proteins accumulate at laser-induced DNA damage sites. They also sought to assess the reversibility of NHEJ complex assembly. Another objective was to investigate the role of Ku70/80 and XRCC4 in the formation of repair complexes. The study tested whether XRCC4/ligase IV recruitment depends on Ku70/80. The researchers also aimed to detect direct interactions between Ku70 and XRCC4. Their broader aim was to clarify the core components of the NHEJ reaction. This work provides insights into how DNA repair proteins dynamically interact during the repair process.

The researchers used a pulsed near-infrared laser to induce DNA double-strand breaks in living cells. This method allowed them to create DNA damage without visible nuclear disruption. Fluorescence microscopy was used to track the accumulation of NHEJ proteins at the damage sites. Time-lapse imaging captured the kinetics of protein recruitment and removal. The team measured the levels of Ku70/80 and XRCC4/ligase IV at the irradiated areas. They tested the dependency of XRCC4/ligase IV accumulation on Ku70/80. To assess the role of DNA-PK(CS), they compared protein accumulation in its presence and absence. A direct interaction between Ku70 and XRCC4 was detected using biochemical assays.

Laser-induced DNA breaks did not cause visible nuclear damage. NHEJ proteins accumulated at the irradiated areas over time. The levels of Ku70/80 and DNA breaks decreased as repair progressed. Ku heterodimers on DNA ends were in dynamic equilibrium with free Ku70/80. XRCC4/ligase IV accumulation at DNA breaks required Ku70/80 but not DNA-PK(CS). A direct interaction between Ku70 and XRCC4 was observed. This interaction may explain the dependency of XRCC4 recruitment on Ku70/80. The findings suggest that Ku70/80 and XRCC4 form a core NHEJ complex.

The study shows that NHEJ complex assembly is dynamic and reversible in living cells. Ku70/80 and XRCC4 form a flexible tether in the repair process. XRCC4 recruitment depends on Ku70/80 but not DNA-PK(CS). The direct interaction between Ku70 and XRCC4 supports this dependency. These findings suggest that the Ku70/80 and XRCC4/ligase IV complex is central to NHEJ. The dynamic nature of Ku heterodimers on DNA ends is a key feature of the repair process. The authors propose that this core complex facilitates the tethering of DNA ends during repair. The results provide a clearer picture of how NHEJ proteins function together in vivo.