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Originally published In Press as doi:10.1074/jbc.M100389200 on June 29, 2001

J. Biol. Chem., Vol. 276, Issue 37, 35165-35175, September 14, 2001
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The delta  and delta ' Subunits of the DNA Polymerase III Holoenzyme Are Essential for Initiation Complex Formation and Processive Elongation*

Min-Sun SongDagger , Phuong T. Pham§, Matthew OlsonDagger , Jeffrey R. CarterDagger , Mary Ann FrandenDagger , Roel M. Schaaper§, and Charles S. McHenryDagger

From the Dagger  Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262 and the § Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

delta and delta ' are required for assembly of the processivity factor beta 2 onto primed DNA in the DNA polymerase III holoenzyme-catalyzed reaction. We developed protocols for generating highly purified preparations of delta  and delta '. In holoenzyme reconstitution assays, delta ' could not be replaced by delta , tau , or gamma , even when either of the latter were present at a 10,000-fold molar excess. Likewise, delta  could not be replaced by delta ', tau , or gamma . Bacterial strains bearing chromosomal knockouts of either the holA(delta ) or holB(delta ') genes were not viable, demonstrating that both delta  and delta ' are essential. Western blots of isolated initiation complexes demonstrated the presence of both delta  and delta '. However, in the absence of chi psi and single-stranded DNA-binding protein, a stable initiation complex lacking delta delta ' was isolated by gel filtration. Lack of delta -delta ' decreased the rate of elongation about 3-fold, and the extent of processive replication was significantly decreased. Adding back delta -delta ' but not chi psi , delta , or delta ' alone restored the diminished activity, indicating that in addition to being key components required for the beta  loading activity of the DnaX complex, delta delta ' is present in initiation complex and is required for processive elongation.

* This work was supported by National Institutes of Health Research Grant GM35695.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.

To whom correspondence should be addressed.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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