Abstract
The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, tau and gamma, were purified from extracts of strains carrying overexpressing plasmids mutated in their frameshifting sequences such that they produced only one subunit or the other. The purified subunits were used to reconstitute the tau and gamma complexes which were characterized by functional assays. The gamma complex-reconstituted holoenzyme required a stoichiometric excess of DNA polymerase III core, beyond physiological levels, for activity. The tau subunit stimulated the gamma complex 2-fold, but could not be used to reconstitute a holoenzyme with gamma complex and stoichiometric quantities of core. In the presence of adenosine 5'-O-(3'-thiotriphospate) (ATP gamma S), the DNA polymerase III holoenzyme behaves as an asymmetric dimer; it can form only initiation complexes with primed DNA in one-half of the enzyme (Johanson, K. O., and McHenry, C. S. (1984) J. Biol. Chem. 259, 4589-4595). An asymmetric distribution of two products of the dnaX gene, gamma and tau, has been postulated to underlie the asymmetry of holoenzyme. To provide a direct test for this hypothesis, we reconstituted holoenzyme containing only the gamma or tau DnaX proteins. We observed that, although gamma could function in the presence of ATP and high concentrations of DNA polymerase III core, it was nearly inert in the presence of ATP gamma S. In contrast, tau-containing holoenzyme behaved exactly like native holoenzyme in the presence of ATP gamma S. These results implicate tau as a key component required to reconstitute holoenzyme with native behavior and show that tau plays a key role in initiation complex formation. These results also show that gamma is not a necessary component, since all of the known properties of native holoenzyme can be reproduced with a 9-subunit tau-holoenzyme.
Highlights
The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, and ␥, were purified from extracts of strains carrying overexpressing plasmids mutated in their frameshifting sequences such that they produced only one subunit or the other
The and ␥ subunits of DNA polymerase III holoenzyme were purified from overexpressing plasmids which produce only the or ␥ subunits (Figs. 1–3; Tables I and II) in order to investigate the functions and physical properties of each subunit alone, without interference from the other
The purified and ␥ subunits were used to reconstitute the complex (␦␦Ј) and ␥ complex (␥␦␦Ј) (Dallmann and McHenry, 1995). These complexes were mixed with polymerase III core and the  subunit in order to test the hypothesis that an asymmetric distribution of and ␥ in holoenzyme (Hawker and McHenry, 1987) was responsible for the asymmetric activity of holoenzyme when assayed with adenosine 5-O-(3-thiotriphospate) (ATP␥S) (Fig. 5C) (Johanson and McHenry, 1984)
Summary
Plasmid pJF119EH was obtained from Peter Furste (Furste et al, 1986) and pDM1 was obtained from Mead et al (1985). SP-Sepharose Ion Exchange Chromatography—One-half of fraction II pellets were dissolved in buffer SP and diluted to a conductivity of 50 mM NaCl (226 ml) This material was loaded onto a 900-ml SP Sepharose (Pharmacia Biotech Inc., 7.5 ϫ 20 cm) column equilibrated with buffer SP. Q-Sepharose Chromatography—One-half of fraction II pellets were dissolved in buffer SP and diluted to a conductivity of 50 mM NaCl (244 ml) This material was loaded onto a 900-ml Q Sepharose (Pharmacia, 7.5 ϫ 20 cm) column equilibrated in buffer SP. Assays of the and ␥ subunits contained 4 pmol of core (unless otherwise stated), 400 fmol of , 540 pmol of M13Gori (as nucleotide), 500 fmol each of ␦, ␦Ј, and ␥, 60 units of DnaG primase, and 1.6 g of E. coli single-stranded DNA binding protein (SSB). The extinction coefficient for (monomer) was determined to be ⑀280 ϭ 42,577 liters molϪ1 cmϪ1, and that for ␥ (monomer), ⑀280 ϭ 25,216 liters molϪ1 cmϪ1
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