Fig. 1

Ku and X4L4 can mediate efficient noncovalent synapsis of blunt dsDNA ends. a Schematic of the smFRET assay for blunt end synapsis. An 85 bp duplex with a terminal 5′-OH and a Cy5 dye located 4 bp away from the end was immobilized on the surface of the imaging coverslip. The 74 bp incoming dsDNA with a terminal 5′-OH and a Cy3 dye located 4 bp away from the end was added to the reaction via an injected solution that also contained 25 nM Ku and 50 nM X4L4. The figure illustrates a lateral configuration that may permit movement of the duplexes along one another [rather than an end-to-end configuration (see below)]. b Representative single-molecule time traces of donor (green) intensity, acceptor (magenta) intensity, and the corresponding E_FRET values (blue) for synapsis mediated by Ku and X4L4. Histograms of donor intensity (green), acceptor intensity (magenta), and E_FRET values (blue) within synapsis period are indicated at the right part of the figure. c Left panel: representative trace for dwell time (t_synapsis) calculation. Right panel: histogram and corresponding exponential fit of total synapsis time mediated by Ku and X4L4. Cy3 signal lifetime (including zero-FRET and detectable FRET portions) of the synaptic complex was used to calculate the total dwell time for each synapsis event and only the synapsis events (n = 139) with both start and end time points within the detection time window were included. Synapsis time shown on graph is represented as mean ± SD of two replicates. d Histogram of E_FRET values of all synapsis events mediated by Ku and X4L4. The E value shown on the graph was obtained from a Gaussian fit of the highest peak. n = 515 molecules. e One of the dynamic intensity traces (donor: green; acceptor: magenta) and corresponding E_FRET values (blue) of synapsis mediated by Ku and X4L4. The black line represents the smoothed trace of corresponding donor, acceptor, or E_FRET trace. Source data are provided as a Source Data1 file