Figure 2.

Effect of complex end structures on pairing dynamics of single molecule complexes with LIG4^WT or LIG4^ΔI (A) smFRET NHEJ assay: (1) dsDNA with a Cy5 acceptor is tethered to a bitonylated PEG surface via a biotin-neutravadin linkage, (2) dsDNA with a Cy3 donor and NHEJ proteins (green) are added to the chamber, and (3) ends are paired and FRET is observed. (B) Quantitation of pairing efficiency of ends with complementary (G:C) or mismatched (GxT, GxA) overhangs by Ku, XLF, XRCC4 and either LIG4^WT (gray) or LIG4^ΔI (orange). Error bars represent standard error of the mean for 3 experiments. Means were assessed by two-way ANOVA as significantly different from control (LIG4^WT on G:C substrate) with confidence p<0.001 (***). (C) Histograms of observed E_FRET for PECs formed as in (B). (D) Representative smFRET trajectory for LIG4^WT and LIG4^ΔI PECs formed with GxT ends demonstrating altered transition frequency and FRET states (E) LIG4^WT enables PECs to oscillate between high and low E_FRET states in response to distortions, and this flexibility is essential for joining distorted breaks.