T7 gene 2.5 ssDNA-binding protein (gp2.5) plays a key role in T7 DNA replication, recombination, and repair. It interacts with T7 DNA polymerase and T7 helicaselprimase, and is essential for coordination of leading and lagging strand synthesis. Gp2.5 stimulates the primase activity of gp4 and the polymerase activity of T7 potymerase.Biochemical and genetic studies have identified the acidic C-termi-nus of gp2.5 as a region involved in interactions with T7 DNA polymerase, T7 DNA helicase/primase and itself to form a dimer. In addition the C-terminus modulates the ssDNA-binding activity of the gp2.5 protein. The goal of this proposal is to understand the role of the C-terminus of gp2.5 in the coordination of protein and ssDNA interactions within the T7 replisome. Using crosslinking, proteolysis, and mass spectrometry we plan to identify the contact points between gp2.5 and gp4 (T7 helicasetprimase), gp2.5 and gp5 (T7 polymerase), and the packing of the gp2.5 C-terminus with respect to the rest of the molecule. Site-directed mutagenesis, affinity chromatography, fluorescence spectroscopy, and surface plasmon resonance will be used to study these interactions in detail. The proposed studies will contribute to our understanding of the mechanisms of coordination of the various enzymatic activities at the replication fork and provide a starting point for understanding the process in more complex systems.
