Reconstitution of a Minimal DNA Replicase from Pseudomonas aeruginosa and Stimulation by Non-cognate Auxiliary Factors

Abstract

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 (␤), single-stranded DNA-binding protein, a complex containing the polymerase (␣) and exonuclease (⑀) subunits, and the essential components of the DnaX complex ( 3 ␦␦). Efficient primer elongation requires the presence of ␣⑀, ␤, and 3 ␦␦. Pseudomonas aeruginosa ␣⑀ can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas ␤ and 3 ␦␦ 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 subunit was not apparent in sequence similarity searches, addition of purified E. coli and (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas 3 ␦␦ complex ϳ10-fold and enables the reconstituted enzyme to function better under physiological salt conditions.

Conservation of Eubacterial Replicases

“…This enzyme possesses its own proofreading activity, is highly processive, and is essential in organisms containing the gene. The interaction of PolC with t would appear to be weaker than the binding between the a and t subunits (4,8,9), suggesting that t does not secure this polymerase as tightly to the replisome. In E. coli, it appears that the t subunit might sequester the a subunit from b 2 until it 'senses' the correct DNA context for its binding to b 2 (12).…”

“…2, Schaeffer et al (1)). Orthologues of the d' and t/g subunits have been easily identified in all eubacteria, but it is only very recently that orthologues of the d subunit have been recognized in eubacteria outside of the gamma and beta subdivisions of the proteobacteria (4,7,8). Indeed, although the d subunit is required for loading of b 2 onto DNA, it is the least conserved of the essential components of the Pol III holoenzyme.…”

“…Recent work from Jarvis and colleagues (8) has demonstrated increased proficiency of the P. aeruginosa holoenzyme and clamp loader functions in the presence of E. coli w and c subunits, and suggest that this indicates that similar subunits may exist in Pseudomonas.…”

“…The assembly of the d, d' and g/t subunits into clamp loaders has been demonstrated for an expanding number of organisms -Streptococcus pyogenes and Staphylococcus aureus, both Gram positive organisms (4,9), the two Gram negative organisms, Helicobacter pylori (Kongsuwan, unpublished) and Pseudomonas aeruginosa (8), and two thermophiles, Thermus thermophilus (6) and Aquifex aeolicus (5). The distribution of the g subunit (a truncated version of t which is also encoded by the dnaX gene) is variable.…”

Discovery and Characterization of the Cryptic Ψ Subunit of the Pseudomonad DNA Replicase

“…We previously reconstituted a minimal DNA replicase from PA consisting of ␣⑀, ␤, and 3 ␦␦Ј (42). Although the reconstituted replicase lacked the , ␥, , and subunits found in E. coli pol III holoenzyme, it exhibited many of the expected characteristics of a replicative holoen-zyme when assayed under low salt conditions in the presence of SSB.…”