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DnaX Complex of Escherichia coli DNA Polymerase III Holoenzyme

1995; Elsevier BV; Volume: 270; Issue: 49 Linguagem: Inglês

10.1074/jbc.270.49.29555

1083-351X

H. Garry Dallmann, Roberta L. Thimmig, Charles S. McHenry,

Antibiotic Resistance in Bacteria

The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, τ and γ, 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 τ andγ complexes which were characterized by functional assays. The γ complex-reconstituted holoenzyme required a stoichiometric excess of DNA polymerase III core, beyond physiological levels, for activity. The τ subunit stimulated theγ complex 2-fold, but could not be used to reconstitute a holoenzyme with γ complex and stoichiometric quantities of core. In the presence of adenosine 5′-O-(3′-thiotriphospate) (ATPγ 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, γ and τ, has been postulated to underlie the asymmetry of holoenzyme. To provide a direct test for this hypothesis, we reconstituted holoenzyme containing only the γ or τ DnaX proteins. We observed that, although γ could function in the presence of ATPγ and high concentrations of DNA polymerase III core, it was nearly inert in the presence of ATPγ S. In contrast, τ -containing holoenzyme behaved exactly like native holoenzyme in the presence of ATPγ S. These results implicate τ as a key component required to reconstitute holoenzyme with native behavior and show that τ plays a key role in initiation complex formation. These results also show that γ is not a necessary component, since all of the known properties of native holoenzyme can be reproduced with a 9-subunit τ -holoenzyme. The alternative forms of the DnaX protein found in Escherichia coli DNA polymerase III holoenzyme, τ and γ, 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 τ andγ complexes which were characterized by functional assays. The γ complex-reconstituted holoenzyme required a stoichiometric excess of DNA polymerase III core, beyond physiological levels, for activity. The τ subunit stimulated theγ complex 2-fold, but could not be used to reconstitute a holoenzyme with γ complex and stoichiometric quantities of core. In the presence of adenosine 5′-O-(3′-thiotriphospate) (ATPγ 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, γ and τ, has been postulated to underlie the asymmetry of holoenzyme. To provide a direct test for this hypothesis, we reconstituted holoenzyme containing only the γ or τ DnaX proteins. We observed that, although γ could function in the presence of ATPγ and high concentrations of DNA polymerase III core, it was nearly inert in the presence of ATPγ S. In contrast, τ -containing holoenzyme behaved exactly like native holoenzyme in the presence of ATPγ S. These results implicate τ as a key component required to reconstitute holoenzyme with native behavior and show that τ plays a key role in initiation complex formation. These results also show that γ is not a necessary component, since all of the known properties of native holoenzyme can be reproduced with a 9-subunit τ -holoenzyme.