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Originally published In Press as doi:10.1074/jbc.M110198200 on February 21, 2002

J. Biol. Chem., Vol. 277, Issue 19, 17334-17348, May 10, 2002
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Analysis of a Multicomponent Thermostable DNA Polymerase III Replicase from an Extreme Thermophile*

Irina BruckDagger , Alexander Yuzhakov§, Olga Yurieva§, David Jeruzalmi§, Maija SkangalisDagger §, John KuriyanDagger §, and Mike O'DonnellDagger §||

From § The Rockefeller University and Dagger  Howard Hughes Medical Institute, New York, New York 10021

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 tau delta delta ' complex. The dnaN gene encoding the beta  clamp was identified, and the tau delta delta ' 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 °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.

* This work was supported in part by National Institutes of Health Grants GM R01 38839 (to M. O. D.) and GM 45547 (to J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Vertex Pharmaceuticals, 130 Waverly St., Cambridge, MA 02139.

|| To whom correspondence should be addressed: The Rockefeller University and Howard Hughes Medical Institute, 1230 York Ave., New York, NY 10021, Tel.: 212-327-7255; Fax: 212-327-7253; E-mail: odonnel@rockvax.rockefeller.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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