RESUMO
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 delta and delta'. In holoenzyme reconstitution assays, delta' could not be replaced 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 delta', tau, or gamma. Bacterial strains bearing chromosomal knockouts of either the holA(delta) or holB(delta') genes were not viable, demonstrating that 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 chipsi and single-stranded DNA-binding protein, a stable initiation complex lacking deltadelta' was isolated by gel filtration. Lack of delta-delta' decreased the rate of elongation about 3-fold, and the extent of processive replication was significantly decreased. Adding back delta-delta' but not chipsi, delta, or delta' alone restored the diminished activity, indicating that in addition to being key components required for the beta loading activity of the DnaX complex, deltadelta' is present in initiation complex and is required for processive elongation.
