Double-strand breaks (DSBs) are the most severe type of DNA damage, as unrepaired DSBs can lead to genomic instability, malignant tumors, and even cell death. Therefore, investigating the specific repair processes of DSBs is crucial. DSBs exhibit a certain degree of mobility, with ongoing intense debate regarding whether their movement is directional or random[1], [2], [3], [4], and whether DSBs cluster or maintain relative positional stability in mammalian cells[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. On one hand, maintaining the stability of DSB positions can reduce chromosomal translocations, thereby helping to prevent the onset and progression of tumorigenesis[13], [14], [15], [16]. On the other hand, while DSB clustering can enhance repair accuracy and facilitate the formation of the D compartment, it also increases the risk of chromosomal translocations[7], [8], [10], [11].

53BP1 is a pivotal signaling protein in the DNA damage response (DDR) pathway[17], [18], and is commonly used as a marker to characterize DSBs[6], [15], [19], [20], [21]. It also exhibits the property of liquid-liquid phase separation (LLPS)[11], [20], [21], creating DNA repair compartments for DSBs[21]. It is conceivable that adjacent 53BP1 foci are more likely to cluster and merge due to their surface tension[10], although this correlation remains unverified. The presence of surface tension also imposed a size limit on 53BP1 foci[6], [20]. Hence, investigating the relationship between DSB clustering and spatial distance is essential.

The DDR has different functions as it progresses over time[22], [23], [24]. DSB clustering is associated with the ATM and MRN complexes that detect DSBs, highlighting its significance in DDR[8], [11]. However, the temporal properties of DSB clustering during different phases of DDR have never been systematically explored, nor has the influence of clustering at different stages on repair processes been investigated. Currently, the most commonly used method for studying DSB clustering involves inducing site-specific DNA damage with endonucleases, followed by sequencing of these damage sites[8], [10], [11]. However, this method does not give a clear observation of DSB clustering. Moreover, variations in the timing of enzyme expression and their subsequent entry into the nucleus can significantly affect the induction time and dynamics of DSB repair[25].

Laser microirradiation offers several advantages over traditional irradiation or endonuclease techniques[25]: 1. It allows for highly precise damage to the cells, with a resolution comparable to that of microscopy imaging; 2. It enables real-time observation of the dynamic process of early DSB clustering.

In this study, we used the Micropoint for laser microirradiation on cells and demonstrated that non-specifically induced 53BP1 foci cluster. By measuring changes in clustering intensity at different distances, we identified at least two factors that influence this process. Furthermore, clustering occurs during the early phase of the DDR and the repair phase, but not during the repair plateau phase. Clustering at different phases has different implications for DNA repair. Clustering accelerates the DSB repair process. This study provides evidence supporting DSB clustering and explores its temporospatial dynamics.