Reconstitution of a minimal DNA replicase from Pseudomonas aeruginosa and stimulation by non-cognate auxiliary factors.
DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (beta), single-stranded DNA-binding protein, a complex containing the polymerase (alpha) and exonuclease (epsilon) subunits, and the essential components of the DnaX complex (tau(3)deltadelta'). Efficient primer elongation requires the presence of alphaepsilon, beta, and tau(3)deltadelta'. Pseudomonas aeruginosa alphaepsilon can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas beta and tau(3)deltadelta' exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli psi subunit was not apparent in sequence similarity searches, addition of purified E. coli chi and psi (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas tau(3)deltadelta' complex approximately 10-fold and enables the reconstituted enzyme to function better under physiological salt conditions.
Jarvis
Thale C
TC
Replidyne, Inc., Louisville, Colorado 80027, USA.
Beaudry
Amber A
AA
Bullard
James M
JM
Janjic
Nebojsa
N
McHenry
Charles S
CS
D001671:Biochemistry / Q000379:methods; D002847:Chromatography, Agarose; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004258:DNA Polymerase III / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005822:Genetic Vectors; D009841:Oligonucleotides / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004247:DNA; C023140:DNA replicase; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase