The delta and delta ' subunits of the DNA polymerase III holoenzyme are essential for initiation complex formation and processive elongation

delta and delta' are required for assembly of the processivity factor beta (2) onto primed DNA in the DNA polymerase III holoenzyme-catalyzed reaction . We developed protocols for generating highly purified preparations of del ta and delta'. In holoenzyme reconstitution assays, delta' could not be rep laced by delta, tau, or gamma, even when either of the latter were present at a 10,000-fold molar excess. Likewise, delta could not be replaced by del ta', tau, or gamma. Bacterial strains bearing chromosomal knockouts of eith er the holA(delta) or holB(delta') genes were not viable, demonstrating tha t both delta and delta' are essential. Western blots of isolated initiation complexes demonstrated the presence of both delta and delta'. However, in the absence of chi psi and single-stranded DNA-binding protein, a stable in itiation complex lacking delta delta' was isolated by gel filtration. Lack of delta-delta' decreased the rate of elongation about 3-fold, and the exte nt of processive replication was significantly decreased. Adding back delta -delta' but not chi psi, delta, or delta' alone restored the diminished act ivity, indicating that in addition to being key components required for the beta loading activity of the DnaX complex, delta delta' is present in init iation complex and is required for processive elongation.