Protein interactions with DNA are of great importance since they govern many cellular processes. While strong interactions, in general, are easy to find, weak ones often remain overlooked. To overcome this limitation, we have designed a three-component system for the detection of DNA interaction partners based on proximity labeling with biotin. The core element of the three-component system is F-DNA, a DNA sequence containing three specific sites: the DNA sequence of interest, and the sequences for binding of either of the other two components of the system, the DNA-binding promiscuous biotin ligase Tus-TurboID, and the DNA-binding internal control of the GAL4-sfGFP system. Thus, the interaction partners of the DNA sequence of interest and GAL4-sfGFP are colocalized with the biotin ligase Tus-TurboID. After biotin addition, Tus-TurboID biotinylates proximal proteins, which are then isolated and identified. When using the three-component system in vitro, it is necessary to add an appropriate source of proteins, such as cell or tissue lysate, which contains interactions partners of the DNA sequence of interest.
In our work, we present the preparation of the three-component system for in vitro use, the characterization of Tus-TurboID and GAL4-sfGFP in terms of their enzymatic activity and/or interaction with F-DNA, the optimization of reaction conditions for in vitro proximity labeling with biotin, and the validation of the three-component system’s functionality. The latter was successfully achieved with the internal control of the system, GAL4-sfGFP, whereas the second step of the validation, in which we aimed to demonstrate the interaction of the tumor-suppressor protein p53 from the added protein source with the GADD DNA sequence, was not. This was due to the instability of p53 under selected in vitro conditions. In further experiments, it is necessary to find in vitro conditions under which p53 is functional and/or to prove the functionality of the system using another known DNA–protein
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