Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. IV. Reconstitution of an asymmetric, dimeric DNA polymerase III holoenzyme Journal Article


Authors: Wu, C. A.; Zechner, E. L.; Hughes, A. J. Jr; Franden, M. A.; McHenry, C. S.; Marians, K. J.
Article Title: Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. IV. Reconstitution of an asymmetric, dimeric DNA polymerase III holoenzyme
Abstract: Individually purified subunits have been used to reconstitute the action of the Escherichia coli DNA polymerase III holoenzyme (Pol III HE) at a replication fork formed in the presence of the primosome, the single-stranded DNA binding protein, and a tailed form II DNA template. Complete activity, indistinguishable from that of the intact DNA Pol III HE, could be reproduced with a combination of the DNA polymerase III core (Pol III core), the γ · δ complex, and the β subunit. Experiments where the Pol III core in reaction mixtures containing active replication forks was diluted suggested that the lagging-strand Pol III core remained associated continuously with the replication fork through multiple cycles of Okazaki fragment synthesis. Since the lagging-strand Pol III core must dissociate from the 3' end of the completed Okazaki fragment, this suggests that its association with the fork is via protein-protein interactions, lending credence to the idea that it forms a dimeric complex with the leading-strand Pol III core. An asymmetry in the action of the subunits was revealed under conditions (high ionic strength) that were presumably destabilizing to the integrity of the replication fork. Under these conditions, τ acted to stimulate DNA synthesis only when the primase was present (i.e. when lagging-strand DNA synthesis was ongoing). This stimulation was reflected by an inhibition of the formation of small Okazaki fragments, suggesting that, within the context of the model developed to account for the temporal order of steps during a cycle of Okazaki fragment synthesis, the presence of τ accelerated the transit of the lagging-strand Pol III core from the 3' end of the completed Okazaki fragment to the 3' end of the new primer.
Keywords: controlled study; dna binding protein; nonhuman; dna polymerase; dna replication; dna synthesis; protein protein interaction; enzyme activity; dna; escherichia coli; enzyme subunit; glutamic acid; glutamates; dna, bacterial; dna template; enzyme reconstitution; dna primase; ribonucleosides; ionic strength; dna polymerase iii; priority journal; article; electrophoresis, agar gel; support, non-u.s. gov't; support, u.s. gov't, p.h.s.
Journal Title: Journal of Biological Chemistry
Volume: 267
Issue: 6
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 1992-02-25
Start Page: 4064
End Page: 4073
Language: English
PUBMED: 1346785
PROVIDER: scopus
DOI/URL:
Notes: Article -- Source: Scopus
Citation Impact
MSK Authors
  1. Kenneth Marians
    137 Marians
  2. Carol A. Wu
    5 Wu