Single-molecule FRET studies of the T4 bacteriophage gene 32 protein binding along single stranded DNA.
University of Oregon
Chemistry and Biochemistry
The T4 bacteriophage gene 32 protein (gp32) is a critical component of the T4 DNA replication system, which serves as a model of DNA replication in higher organisms such as humans. Gp32 is a single stranded (ss)DNA binding protein which binds nonspecifically to the ssDNA that is exposed during replication. This serves to protect the ssDNA from nucleases, melts out any secondary structure that would be detrimental to replication, and configures the ssDNA for use by the other proteins in the replication system. Gp32 has been studied for many years, however the mechanism and dynamics of its interactions with ssDNA remain to be understood. We have used single-molecule F'rster Resonance Energy Transfer (FRET) to probe gp32 binding at different salt and gp32 concentrations as well as on different lengths of ssDNA. These studies have revealed repetitive fluctuations in the FRET signal corresponding to the binding and dissociation of gp32 which are dependent on the conditions used. From these fluctuations we have made a molecular interpretation of the cooperative binding of gp32.