Document Type
Article
Publication Date
12-9-2005
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.
Recommended Citation
Jarvis, Thale C.; Beaudry, Amber A.; Bullard, James M.; Ochsner, Urs; Dallmann, H. Garry; and McHenry, Charles S., "Discovery and Characterization of the Cryptic Psi Subunit of the Pseudomonad DNA Replicase" (2005). Chemistry Faculty Publications and Presentations. 25.
https://scholarworks.utrgv.edu/chem_fac/25
First Page
40465
Last Page
40473
Publication Title
Journal of Biological Chemistry
DOI
10.1074/jbc.M508310200
Comments
© 2005 American Society for Biochemistry and Molecular Biology Inc. Original published version available at http://doi.org/10.1074/jbc.M508310200.
Jarvis, T., Beaudry, A., Bullard, R., et al. Discovery and Characterization of the Cryptic Psi Subunit of the Pseudomonad DNA Replicase. Journal of Biological Chemistry 2005, vol. 280, 40465-40473.