DNA Polymerase I 11 Holoenzyme of Escherichia coli 11 . A NOVEL COMPLEX INCLUDING THE y SUBUNIT ESSENTIAL FOR PROCESSIVE SYNTHESIS

Processive DNA synthesis, a property of DNA polymerase I11 holoenzyme of Escherichia coli, was not achieved by combining the pol I11 core (a, e, and B subunits) and the 8 and y 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 7 and y subunits. Purified to homogeneity, the activity, assayed by reconstitution of processivity, was represented by five polypeptides which were copurified. Judged by sodium dodeeyl sulfate-polyacrylamide gel electrophoresis, these correspond to the known subunits y (52 kDa) and 6 (35 kDa) and to three new polypeptides: 6‘ (33 kDa), x (15 kDa), and $ (12 kDa). The five polypeptides form a tight complex with a native molecular weight of about 200 kDa and a subunit stoichiometry of two 7 subunits to one each of the others. Processive DNA synthesis, now achieved with only three components (pol I11 core, 8, and the auxiliary complex), provides the opportunity to assess the functions of each and the contribution that the remaining auxiliary 7 subunit makes to reconstitute a holoenzyme.