Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains

The and proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with being a truncated version of arising from ribosomal frameshifting. is comprised of five structural domains, the first three of which are shared by (Gao, D., and McHenry, C. (2001) J. Biol. Chem. 276, 4433– 4453). In the absence of the other holoenzyme subunits, DnaX exists as a tetramer. Association of , , , and with domain III of DnaX4 results in a DnaX complex with a stoichiometry of DnaX3 . To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V. Unlike domain I-II, treatment of domain III-V, , and with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein. Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer. Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting , , , , and onto streptavidin-agarose beads. Thus, domain III not only contains the , , , and binding interface, but also the region that enables DnaX to oligomerize.