DnaX complex of Escherichia coli DNA polymerase III holoenzyme. Physical characterization of the DnaX subunits and complexes.

Abstract:

A physical characterization of the tau and gamma subunits of the Escherichia coli DNA polymerase III holoenzyme and their complexes with the delta, delta', chi, and psi subunits is presented. The native molecular mass of the tau and gamma subunits was determined to be 255,000 and 189,000 Da, respectively, by sedimentation equilibrium analytical ultracentrifugation. Both values indicate a tetrameric quaternary structure. The tau and gamma complexes were reconstituted and purified using two different methods. Both complexes assembled readily and were reconstituted at subunit concentrations approaching physiological levels. The stoichiometries of the tau and gamma complexes, as determined by quantitative densitometry of SDS-polyacrylamide gels, were found to be tau 4 delta 1 delta' 1 chi 1 psi 1 and gamma 4 delta 1 delta' 1 chi 1 psi 1. BIAcore analysis demonstrated that the formation of large multiprotein complexes of holoenzyme subunits depends on the presence of the tau subunit; gamma could not substitute. We present a model for a gamma-less form of DNA polymerase III holoenzyme that has asymmetrical structural features that may be responsible for the functional asymmetry observed in holoenzyme. The stoichiometry of the reconstituted DNA polymerase III* component of holoenzyme in this model is (alpha epsilon theta)2DnaX4 delta 1 delta' 1 chi 1 psi 1.

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