tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX.
The tau and gamma proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with gamma being a truncated version of tau arising from ribosomal frameshifting. tau is comprised of five structural domains, the first three of which are shared by gamma (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 delta, delta', chi, and psi with domain III of DnaX(4) results in a DnaX complex with a stoichiometry of DnaX(3)deltadelta'chipsi. 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, gamma, and tau 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 tau, delta, delta', chi, and psi onto streptavidin-agarose beads. Thus, domain III not only contains the delta, delta', chi, and psi binding interface, but also the region that enables DnaX to oligomerize.
Glover
B P
BP
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Pritchard
A E
AE
McHenry
C S
CS
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001704:Biopolymers; D004258:DNA Polymerase III / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016875:tau Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D001704:Biopolymers; C103076:DnaX protein, Bacteria; D011993:Recombinant Fusion Proteins; D016875:tau Proteins; D004258:DNA Polymerase III