Fig. 1: SMADNE analysis of HaloTag635-LIG3α-XRCC1-YFP on eight observable nicks in λ DNA.

a A representative structural model of heterodimeric complex model of N-terminal tagged HaloTag-635-LIG3α and C-terminal YFP tagged XRCC1 bound to nicked DNA (PDB codes 3L2P and 3K77) using Alphafold 3, for clarity the disordered regions were removed. b Domain structures of LIG3α (nuclear) bound to C-terminal BRCT2 domain of XRCC1 via BRCT domain, forming a heterodimeric complex. c A schematic 3D model of workflow showing DNA-binding interactions of heterodimeric complex model of N-terminal tagged HaloTag-635-LIG3α and C-terminal YFP tagged XRCC1 bound to nicked λ DNA digested with nicking enzyme Nt.BspQI generating eight observable ligatable nicks. The substrate (48.5 kb nicked DNA) then gets attached between two polystyrene beads, trapped in the optical tweezers shown as a 2D scan image captured with the binding events for HaloTag-635-LIG3α-XRCC1-YFP followed by real time kymograph mode at 10 pN tension, where X-axis represents time (sec), and the Y-axis shows the position (μm). d Cumulative residence time distribution (CRTD) and cumulative gap time distribution (CGTD) analysis for HaloTag-635-LIG3α binding nicked DNA, with a single exponential fit shown in red. Asterisks indicate a single binding event occurring in real time. e Cumulative residence time distribution (CRTD) and cumulative gap time distribution (CGTD) analysis for XRCC1-YFP binding nicked DNA, with a single exponential fit shown in blue. Asterisks indicate a single binding event occurring in real time. f 11 possible interactions for HaloTag-635-LIG3α as red bars and XRCC1-YFP as blue bars binding on DNA respectively. g The distribution of the 11 categories for HaloTag-635-LIG3α and XRCC1-YFP binding nicked λ DNA. Error bars represent the SEM of fourteen experiments. Created in BioRender. Gupta, A. (2025) https://BioRender.com/3hbm9as. Source data are provided as a Source Data file.