Figure 4.

NEIL3 Zf-GRF repeat has higher binding affinity to Cy5-labeled 70-nt ssDNA than NEIL3-ZF1 using MST assays. A, a diagram showed the overall steps and conditions used for MST experiments including the structure of substrate (target) and structure of each of four ligands. B, binding curves constructed for NEIL3-ZF1&2 (n = 4), NEIL3-ZF1 (n = 3), and NEIL3-ZF1&2 K553A (n = 3) with GST (n = 3) as control using 50 nm Cy5-labeled ssDNA in Buffer A. K_d value for GST-NEIL3-ZF1&2 and GST-NEIL3-ZF1 was 3.9 ± 0.55 and 7.9 ± 0.68 μm, respectively. F_norm is normalized fluorescence measured by F₁ as fluorescence after thermodiffusion (at 4-5 s range) divided by F₀ as initial fluorescence.