Implementing the Bimolecular Fluorescence Complementation Assay to Study Protein Interactions in the Cell Cycle Checkpoint Response

Abstract

The genomic integrity of a cell is constantly being pressured by both intrinsic and extrinsic forces. Cell cycle checkpoints exist to protect the cells by arresting cell cycle progression in response to DNA damage or replication stress. It has been shown that the interaction between the checkpoint proteins Rad9A and TopBP1 is a crucial upstream event required for the ATR-dependent checkpoint response to DNA damage, which can be activated throughout different points in the cell cycle. The Bimolecular Fluorescence Complementation (BiFC) technique has recently emerged as a simple and effective tool for analyzing protein-protein interactions in live cell cultures. By fusing complementary fragments of fluorescent proteins to proteins of interest, one can visualize protein-protein interactions through the formation of a mature fluorophore from these fragments. In the current work, the BiFC assay system was employed to study the interaction between TopBP1 and Rad9A; the human homologue of fission yeast Rad9. BiFC vectors expressing TopBP1, Rad9A, and the Rad9A-S387 mutant were constructed and optimized for transfection in HeLa cells. It was shown that the BiFC fusion protein of Rad9A lacked phosphorylation on its constitutive S387 site, although it retained its upstream damage dependent S272 phosphorylation after IR treatment. BiFC signals could be detected in cells containing the BiFC fusion proteins of Rad9A and TopBP1 using confocal microscopy and flow cytometry techniques. However, the signals could not be distinguished from that of the negative control samples. Our results suggest a possibility that our BiFC fusion proteins of interest interact in a non-specific manner, although further characterization is required to confirm this. The BiFC assay employed in this project must be further optimized to effectively study the interaction between Rad9A and TopBP1, as well as other checkpoint proteins. However, this study has given us great insight into the implementation of this new BiFC technique for studying protein interactions in the context of cell cycle proteins, and the knowledge gained from this study will be invaluable for future work.