Analysis of a multicomponent thermostable DNA polymerase III replicase from an extreme thermophile.
This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus. Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (tau, delta, and delta') were identified. The dnaX gene produces only the full-length product, tau, and therefore differs from Escherichia coli dnaX that produces two proteins (gamma and tau). Nonetheless, the A. aeolicus proteins form a taudeltadelta' complex. The dnaN gene encoding the beta clamp was identified, and the taudeltadelta' complex is active in loading beta onto DNA. A. aeolicus contains one dnaE homologue, encoding the alpha subunit of DNA polymerase III. Like E. coli, A. aeolicus alpha and tau interact, although the interaction is not as tight as the alpha-tau contact in E. coli. In addition, the A. aeolicus homologue to dnaQ, encoding the epsilon proofreading 3'-5'-exonuclease, interacts with alpha but does not form a stable alpha.epsilon complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system. Despite these differences to the E. coli system, the A. aeolicus proteins function to yield a robust replicase that retains significant activity at 90 degrees C. Similarities and differences between the A. aeolicus and E. coli pol III systems are discussed, as is application of thermostable pol III to biotechnology.
Bruck
Irina
I
Rockefeller University and Howard Hughes Medical Institute, New York, New York 10021, USA.
Yuzhakov
Alexander
A
Yurieva
Olga
O
Jeruzalmi
David
D
Skangalis
Maija
M
Kuriyan
John
J
O'Donnell
Mike
M
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; C103076:DnaX protein, Bacteria; D011994:Recombinant Proteins; D004247:DNA; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C083980:dnaN protein, Bacteria