DNA polymerase III holoenzyme of Escherichia coli. II. A novel complex including the gamma subunit essential for processive synthesis.
Processive DNA synthesis, a property of DNA polymerase III holoenzyme of Escherichia coli, was not achieved by combining the pol III core (alpha, epsilon, and theta subunits) and the beta and gamma subunits. An activity that restored processivity to these subunits was found in crude extracts and was overproduced 4-fold in cells with plasmids amplifying the tau and gamma subunits. Purified to homogeneity, the activity, assayed by reconstitution of processivity, was represented by five polypeptides which were copurified. Judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these correspond to the known subunits gamma (52 kDa) and delta (35 kDa) and to three new polypeptides: delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The five polypeptides form a tight complex with a native molecular weight of about 200 kDa and a subunit stoichiometry of two gamma subunits to one each of the others. Processive DNA synthesis, now achieved with only three components (pol III core, beta, and the auxiliary complex), provides the opportunity to assess the functions of each and the contribution that the remaining auxiliary tau subunit makes to reconstitute a holoenzyme.
Maki
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Department of Biochemistry, Stanford University School of Medicine, California 94305.
Kornberg
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D004258:DNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances
D046911:Macromolecular Substances; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase