Core NHEJ Factors Maintain Synapsis Across Diverse DNA End Structures

DNA double strand breaks (DSBs) generated by genotoxic stress frequently contain chemically diverse termini that require end processing before ligation by non-homologous end joining (NHEJ). How end chemistry influences assembly and persistence of NHEJ synaptic complexes remains unclear. Using single-molecule imaging approaches in Xenopus egg extract, we measured formation and stability of the long-range (LRC) and short-range (SRC) complexes on nine DNA substrates spanning kinase-, polymerase- and nuclease-dependent processing requirements. LRC formation and lifetime were comparable across end types, and SRC formation and stability were largely insensitive to end chemistry. DNA hairpins, physiological intermediates of V(D)J recombination, were a notable exception that formed LRCs but inefficiently transitioned to SRCs. Depletion of cognate processing factors had modest effects on SRC formation and persistence, indicating that core NHEJ factors provide the principal structural support for synapsis across diverse termini. Although XLF was required for SRC formation on all substrates, reducing an XLF dimer from two intact tail/Ku-binding motifs to one selectively reduced productive SRC formation on ends requiring processing. Together, these results support a model in which partially redundant protein-protein interactions between core NHEJ factors maintain synapsis while end-specific processing reactions shape repair outcome.