Early Double-Strand Break Signaling is asynchronous and accelerated at complex breaks

DNA double-strand break (DSB) repair foci are dynamic nuclear structures essential for coordinating the cellular response to genomic damage. While their composition and function have been extensively studied, little is known about the early kinetics of individual foci. Here, we present a high-resolution, time-resolved analysis of the formation and maturation of DSB repair foci in living cells using ultra-soft X-ray irradiation combined with 3D time-lapse microscopy. We quantify the sequence, recruitment sequence, and dynamics of key DNA damage response (DDR) proteins (MDC1, RNF8, RNF168, 53BP1) at single-foci resolution, and uncover marked heterogeneity in their onset and maturation. Surprisingly, foci appear asynchronously, with onset of some proteins (e.g., RNF168) consistently breaking the canonical recruitment sequence (MDC1 > RNF8 > RNF168 > 53BP1). We demonstrate that this variability is affected by multiple factors, and notably by DSB complexity, as inferred from linear energy transfer (LET). High-LET radiation consistently accelerated foci onset across multiple experimental setups, implicating DSB complexity as a key driver of DDR kinetics. Collectively, our findings highlight the intricate, context-dependent nature of early DDR signaling and suggest that foci dynamics are shaped by both physical characteristics of DNA lesions and the cellular environment. These insights refine current models of DSB repair and underscore the value of single-lesion analyses in understanding genome maintenance.