DNA replication is catalyzed by a host of enzymes that work together to unwind the DNA and make an accurate copy of each strand. Errors in DNA synthesis can lead to mutations in genomic DNA and the production of malformed proteins, which can disrupt cellular functions and lead to diseases such as cancer. Bacteriophage T7 is an excellent model system for understanding the structural basis for the fidelity, processivity, and mechanism of DNA replication. The entire replication process is encoded by five proteins: the DNA polymerase encoded by gene 5, its processivity factor E. Coli thioredoxin, two subunits of the helicase- primase encoded by gene 4, and the gene 2.5 single-stranded DNA binding protein (gp2.5). The rate of DNA synthesis in vitro is drastically reduced in the absence of gp2.5, and it is essential for DNA replication in vivo, greatly enhancing the processivity of both leading and lagging strand synthesis. The simplicity of the T7 replication system affords an opportunity to effectively study gp2.5's individual role in replication as well as its interactions with the other proteins of the replication fork. The structure of gp2.5 will be investigated through x-ray crystallographic studies of gp2.5 alone and bound to DNA. The structure of the complex will directly reveal the mode of interaction with DNA. Gp2.5 physically interacts with the gene 5 polymerase-thioredoxin complex (the T7 DNA polymerase holoenzyme) and the gene 4 helicase- primase. These interactions are vital for the coupling of leading and lagging strand synthesis. The interaction of gp2.5 and T7 DNA polymerase will be more precisely characterized by analytical ultracentrifugation. Sedimentation equilibrium experiments will directly measure the stability and subunit stoichiometry of this protein-protein association. This information will be used in preparation for the crystallization of the complex. The interactions of gp2.5 and the gene 4 helicase-primase will be investigated through x-ray crystallographic studies. The determination of the structure of the gp2.5-T7 DNA polymerase holoenzyme complex or the gp2.5-gene 4 complex will provide direct information on the interactions between proteins at the replication fork.
