Discovery and characterization of the cryptic psi 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, chi and Psi are tightly associated clamp loader accessory subunits. The addition of E. coli chiPsi to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of chi and Psi 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 chi and Psi were co-expressed and purified as a 1:1 complex. P. aeruginosa chiPsi increased the specific activity of tau(3)deltadelta' 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between chiPsi 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.
Jarvis
Thale C
TC
Replidyne, Inc., Louisville, Colorado 80027, USA. tjarvis@replidyne.com
Beaudry
Amber A
AA
Bullard
James M
JM
Ochsner
Urs
U
Dallmann
H Garry
HG
McHenry
Charles S
CS
D000595:Amino Acid Sequence; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D016415:Sequence Alignment
D021122:Protein Subunits; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase