Abstract

Previously, we identified that both fingers 1 and 2 in the three Cys₂His₂ zinc-finger domains (TZD) of testis zinc-finger protein specifically bind to its cognate DNA; however, finger 3 is non-sequence–specific. To gain insights into the interaction mechanism, here we further investigated the DNA-binding characteristics of TZD bound to non-specific DNAs and its finger segments bound to cognate DNA. TZD in non-specific DNA binding showed smaller chemical shift perturbations, as expected. However, the direction of shift perturbation, change of DNA imino-proton NMR signal, and dynamics on the ¹⁵N backbone atom significantly differed between specific and non-specific binding. Using these unique characteristics, we confirmed that the three single-finger segments (TZD₁, TZD₂ and TZD₃) and the two-finger segment (TZD₂₃) non-specifically bind to the cognate DNA. In comparison, the other two-finger segment (TZD₁₂) binding to the cognate DNA features simultaneous non-specific and semi-specific binding, both slowly exchanged in terms of NMR timescale. The process of TZD binding to the cognate DNA is likely stepwise: initially TZD non-specifically binds to DNA, then fingers 1 and 2 insert cooperatively into the major groove of DNA by semi-specific binding, and finally finger 3 non-specifically binds to DNA, which promotes the specific binding on fingers 1 and 2 and stabilizes the formation of a specific TZD–DNA complex.