τ Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains PARTIAL PROTEOLYSIS OF TERMINALLY TAGGED τ TO DETERMINE CANDIDATE DOMAINS AND TO ASSIGN DOMAIN V AS THE α BINDING DOMAIN

Abstract The τ subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the α subunit. In addition to playing critical roles in the structural organization of the holoenzyme, τ mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged τ proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the τ subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous δ′ subunit, and the third region is near the C-terminal end of the τ-δ′ alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335–345). We propose a five-domain structure for the τ protein. Domains I and II are based on the crystallographic structure of δ′ by Guenther and colleagues. Domains III–V are based on our protease cleavage results. Using this information, we expressed biotin-tagged τ proteins lacking specific protease-resistant domains and analyzed their binding to the α subunit by surface plasmon resonance. Results from these studies indicated that the α binding site of τ lies within its C-terminal 147 residues (domain V).