The DnaX complex of the DNA polymerase holoenzyme assembles the beta(2) processivity factor onto the primed template enabling highly processive replication. The key ATPases within this complex are tau and gamma, alternative frameshift products of the dnaX gene. Of the five domains of tau, I-III are shared with gamma In vivo, gamma binds the auxiliary subunits deltadelta' and chipsi (Glover, B. P., and McHenry, C. S. (2000) J. Biol. Chem. 275, 3017-3020). To localize deltadelta' and chipsi binding domains within gamma domains I-III, we measured the binding of purified biotin-tagged DnaX proteins lacking specific domains to deltadelta' and chipsi by surface plasmon resonance. Fusion proteins containing either DnaX domains I-III or domains III-V bound deltadelta' and chipsi subunits. A DnaX protein only containing domains I and II did not bind deltadelta' or chipsi. The binding affinity of chipsi for DnaX domains I-III and domains III-V was the same as that of chipsi for full-length tau, indicating that domain III contained all structural elements required for chipsi binding. Domain III of tau also contained deltadelta' binding sites, although the interaction between deltadelta' and domains III-V of tau was 10-fold weaker than the interaction between deltadelta' and full length tau. The presence of both delta and chipsi strengthened the delta'-C(0)tau interaction by at least 15-fold. Domain III was the only domain common to all of tau fusion proteins whose interaction with delta' was enhanced in the presence of delta and chipsi. Thus, domain III of the DnaX proteins not only contains the deltadelta' and chipsi binding sites but also contains the elements required for the positive cooperative assembly of the DnaX complex.