Abstract
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 chipsi and single-stranded DNA-binding protein, a stable initiation complex lacking deltadelta' 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 chipsi, 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, deltadelta' is present in initiation complex and is required for processive elongation.
Highlights
High processivities and fast elongation rates are hallmarks of the chromosomal replicases of eukaryotes and prokaryotes [1,2,3]
In the absence of auxiliary subunits, ATP is ineffective in inducing the formation of stable complex dramatically decreasing processivity [15]. pol III (␣⑀) activity is inhibited by high salt concentrations and the E. coli single-stranded DNA-binding protein (SSB) in the absence, but not the presence, of auxiliary subunits [5, 16]
These results indicate that lack of these auxiliary subunits does not significantly affect the tight complex assembly of pol III//2 on primed template
Summary
Vol 276, No 37, Issue of September 14, pp. 35165–35175, 2001 Printed in U.S.A. The ␦ and ␦ Subunits of the DNA Polymerase III Holoenzyme Are Essential for Initiation Complex Formation and Processive Elongation*. 1 The abbreviations used are: pol III, DNA polymerase III (␣⑀); DnaX complex, a functional subassembly containing either or both DnaX proteins ( and/or ␥) with associated ␦, ␦Ј, , and ; DnaX protein, either of two alternative products of the dnaX gene ( or ␥); IC, initiation complex, a stable complex between the DNA polymerase III holoenzyme and primed DNA that is formed upon ATP hydrolysis; SSB, E. coli single-stranded DNA-binding protein; dADPNP, deoxyadenylylimidodiphosphate; -complex, 3␦␦Ј; ␥-complex, ␥3␦␦Ј; BSA, bovine serum albumin; n-IC, an IC that was formed using the native holoenzyme and a 30-mer/M13gori primer-template; -IC, an IC that was formed using pol III, 2, the -complex, and a 30-mer/M13gori primer-template; ␥-IC, an IC that was formed using pol III, 2, the ␥-complex and a 30-mer/M13gori primer-template; IC-[␦␦Ј], an auxiliary subunit-deficient IC that was formed using pol III, 2, 4, ␦␦Ј, gel filtered to remove ␦␦Ј; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; kb, kilobase pair; bp, base pair. Evaluation of the rate and extent of DNA chain elongation revealed that ␦ and ␦Ј are involved in elongation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
