Discovery and characterization of the cryptic ψ subunit of the pseudomonad DNA replicase

We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of alpha and epsilon ( polymerase and editing nuclease), beta( processivity factor), and the essential tau, delta, and delta' components of the clamp loader complex ( Jarvis, T., Beaudry, A., Bullard, J., Janjic, N., and McHenry, C. ( 2005) J. Biol. Chem. 280, 7890-7900). In Escherichia coli DNA polymerase III holoenzyme, alpha and Psi are tightly associated clamp loader accessory subunits. The addition of E. coli chi Psi to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of alpha and beta counterparts in P. aeruginosa. The P. aeruginosa chi subunit was recognizable from sequence similarity, but Psi was not. Here we report purification of an endogenous replication complex from P. aeruginosa. Identification of the components led to the discovery of the cryptic psi subunit, encoded by holD. P. aeruginosa alpha and epsilon were co-expressed and purified as a 1: 1 complex. P. aeruginosa chi psi increased the specific activity of tau(3)delta delta' 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between tau,delta and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa psi allowed us to identify psi subunits from several other Pseudomonads and to predict probable translational start sites for this protein family. This represents the first identification of a highly divergent branch of the psi family and confirms the existence of psi in several organisms in which psi was not identifiable based on sequence similarity alone.