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

• Published in: The Journal of biological chemistry Vol. 267; no. 6; pp. 4064 - 4073
• Main Authors: C A Wu, E L Zechner, A J Hughes, Jr, M A Franden, C S McHenry, K J Marians
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25-02-1992
• Subjects: DNA - metabolism; DNA Polymerase III - metabolism; DNA Replication - drug effects; DNA, Bacterial - biosynthesis; DNA, Bacterial - metabolism; Electrophoresis, Agar Gel; Escherichia coli; Escherichia coli - genetics; Glutamates - pharmacology; Glutamic Acid; Ribonucleosides - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)50631-7

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 gamma.delta complex, and the beta 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, tau 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 tau 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.