Date Published: April 6, 2017
Publisher: Public Library of Science
Author(s): Chun-Chi Chou, Shu-Yi Wei, Yuan-Chao Lou, Chinpan Chen, Michael Massiah.
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.
DNA-binding proteins initiate gene transcriptional regulation by searching for their target DNA sites among an overwhelming number of non-specific DNA sequences in the nucleus. Theoretically, the protein first binds non-specifically to DNA, then rapidly searches the sequence for the presence of specific binding sites . Therefore, protein–DNA recognition includes at least two steps, non-specific and specific binding [2–4].
DNA recognition by classical Cys2His2 zinc-finger proteins is essential for specific regulation of genes. Most studies and comparisons have mainly focused on the free protein as well as the specific protein–DNA complex in examining DNA recognition. Non-specific protein–DNA binding is an important intermediate step in the recognition process, so in this work we present evidence to show that TZD binding to the cognate DNA is stepwise, initially non-specific, then semi-specific, and finally specific.