τ Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains

Abstract

The τ and γ proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with γ being a truncated version of τ arising from ribosomal frameshifting. τ is comprised of five structural domains, the first three of which are shared by γ (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 δ, δ′, χ, and ψ with domain III of DnaX₄ results in a DnaX complex with a stoichiometry of DnaX₃δδ′χψ. 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, γ, and τ 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 τ, δ, δ′, χ, and ψ onto streptavidin-agarose beads. Thus, domain III not only contains the δ, δ′, χ, and ψ binding interface, but also the region that enables DnaX to oligomerize.