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. 2016 Oct 4;113(42):11853–11858. doi: 10.1073/pnas.1606721113

Fig. S5.

Fig. S5.

Calculation of koff and kon. (A and B) ton (A) and toff (B) histograms of RAG1/2AF546 for surface-tethered 12RSS, 23RSS, and dsDNA substrates in the presence of 5 nM RAG1/2, 10 nM HMGB1, and 1 nM RSS (Mg2+). Plots were fit with exponential decay curves to extract binding kinetics. koff and kon were generated using the inverse of ton (1/ton) and toff (1/toff), respectively. (C) Representative single-molecule FRET trajectory for the 12RSS substrate in the presence of 50 nM RAG–HMGB1 complex. (D) Representative single-molecule trajectories for dsDNA substrate in the presence of 5 nM RAG1/2, 10 nM HMGB1, and 1 nM 23RSS (Mg2+). Regardless of cofactors in solution, RAG1/2 has minimal interaction with the non-RSS substrate. (E) Calculated KD for 12RSS, 23RSS, and dsDNA substrates.