Abstract
The protein-induced fluorescence change technique was employed to investigate the interactions between proteins and their DNA substrates modified with the Cy3 fluorophore. It has been reported that the human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal proline-tryptophan-tryptophan-proline (PWWP) domain (the N-terminal 100 amino acids of HDGF) capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancer. This project investigated the specific binding behavior of HDGF, the PWWP domain, and the C140 domain (the C-terminal 140 amino acids of HDGF) sequentially using protein-induced fluorescence change. We found that the binding of HDGF and its related proteins on Cy3-labeled 15 bp SMYD1 dsDNA will cause a significant decrease in the recorded Cy3 fluorophore intensity, indicating the occurrence of protein-induced fluorescence quenching. The dissociation equilibrium constant was determined by fitting the bound fraction curve to a binding model. An approximate 10-time weaker SMYD1 binding affinity of the PWWP domain was found in comparison to HDGF. Moreover, the PWWP domain is required for DNA binding, and the C140 domain can enhance the DNA binding affinity. Furthermore, we found that the C140 domain can regulate the sequence-specific binding capability of HDGF on SMYD1.