Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-018-0065-1 (2018)
Nonhomologous end joining (NHEJ) mediates repair of double-strand DNA (dsDNA) breaks through formation of a multimeric complex. The Ku heterodimer binds DNA ends and recruits the catalytic subunit DNA-PKcs to form the DNA-PK holoenzyme, which bridges two DNA ends. DNA-PK recruits additional factors such as PAXX, XRCC4, XLF and ligase IV to mediate DNA synapsis. To determine the contribution of each component to synapsis stability, Wang et al. utilized a single-molecule forceps approach on a dsDNA construct with two overhanging blunt ends facing each other combined with a dsDNA linker. By modulating force on one end of the DNA construct, DNA synapsis formation could be characterized by observing the change in length of the construct. Using the complete NHEJ complex, the authors observed synaptic junctions with 66-second lifetimes. Loss of a single protein such as PAXX or one of the XRCC4, XLF or ligase IV components reduced synapsis lifetime; however, loss of two proteins nearly abolished synapsis entirely. This suggests that NHEJ complexes progressively assemble and stabilize through the summation of weak yet additive interactions between components. This study provides unique insights into the NHEJ machinery and ushers in a detailed chemomechanical understanding of DNA repair complexes.