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

Abstract

We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of α and ε (polymerase and editing nuclease), β (processivity factor), and the essential τ, δ, and δ′ 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, χ and ψ are tightly associated clamp loader accessory subunits. The addition of E. coli χψ to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of χ and ψ counterparts in P. aeruginosa. The P. aeruginosa χ subunit was recognizable from sequence similarity, but ψ 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 ψ subunit, encoded by holD. P. aeruginosa χ and ψ were co-expressed and purified as a 1:1 complex. P. aeruginosa χψ increased the specific activity of τ3δδ′ 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between χψ and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa ψ allowed us to identify ψ 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 ψ family and confirms the existence of ψ in several organisms in which ψ was not identifiable based on sequence similarity alone. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.