t Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains

Interaction between the t subunit of the DNA polymerase III holoenzyme and the DnaB helicase is critical for coupling the replicase and the primosomal apparatus at the replication fork (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643– 650). In the preceding manuscript, we reported the identification of five putative structural domains within the t subunit (Gao, D., and McHenry, C. (2000) J. Biol. Chem. 275, 4433–4440). As part of our systematic effort to assign functions to each of these domains, we expressed a series of truncated, biotin-tagged t fusion proteins and determined their ability to bind DnaB by surface plasmon resonance on streptavidin-coated surfaces. Only t fusion proteins containing domain IV bound DnaB. The DnaB-binding region was further limited to a highly basic 66-amino acid residue stretch within domain IV. Unlike the binding of immobilized t4 to the DnaB hexamer, the binding of monomeric domain IV to DnaB6 was dependent upon the density of immobilized domain IV, indicating that DnaB6 is bound by more than one t protomer. This observation implies that both the leading and lagging strand polymerases are tethered to the DnaB helicase via dimeric t. These double tethers of the leading and lagging strand polymerases proceeding through the t-t link and an additional t-DnaB link are likely important for the dynamic activities of the replication fork.