Fig. 6

Ensemble confirmation and the proposed model of NHEJ synapsis. a (Top) Gel results of blunt end ligation mediated by different combinations of NHEJ components in the presence and absence of 10% (v/v) PEG-8000. (Bottom) Quantified ligation efficiency. Reactions 1–12 correspond to the reactions represented by lanes 1–12 in the top panel, respectively. Data are represented as mean ± SD of two independent replicates. PEG-8000 is a volume excluder, which increases the collision frequency of the two dsDNA together ready for covalent ligation. With this volume excluder in the solution, the ligation efficiency was similar with or without stimulation from XLF, PAXX, or both (lanes 3–6). Without the volume excluder in the solution, where only NHEJ proteins would be available to bridge the two dsDNA ends for ligation, we find that Ku and X4L4 cannot mediate the ligation of blunt end dsDNA (lane 9), and XLF or PAXX can stimulate the covalent ligation (lanes 10–12). BZ15 was synthesized with a 5′ PO₄. b Ku and X4L4 mediate a flexible synapsis (FS), in which two dsDNA are brought into a lateral configuration. Aligning the two dsDNA within the FS complex to an end-to-end state is stimulated by the XLF or PAXX protein, and XLF is the more efficient one in this respect (as reflected by the length of the reaction arrow). One XLF dimer either interacting with X4 in X4L4 or with Ku may be sufficient for close synapsis (CS), but we speculate that more than one XLF dimer binding to both Ku and X4 may more fully stabilize the CS complex. We have not shown filament formation in this diagram, but for chromatinized DNA templates, it is possible that filaments may be important¹³. Source data are provided as Source Data1 and Source Data2 files