Journal
EMBO JOURNAL
Volume 19, Issue 23, Pages 6536-6545Publisher
WILEY-BLACKWELL
DOI: 10.1093/emboj/19.23.6536
Keywords
DNA polymerase III; DNA replication; multiprotein operon; protein assembly; protein complex
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Funding
- NIGMS NIH HHS [R01 GM035695, R01GM35695] Funding Source: Medline
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We have constructed a plasmid-borne artificial operon that expresses the six subunits of the DnaX complex of Escherichia coli DNA polymerase III holoenzyme: tau, gamma, delta, delta', chi and psi. Induction of this operon followed by assembly in vivo produced two tau gamma mixed DnaX complexes with stoichiometries of tau (1)gamma (2)delta delta'chi psi and tau (2)gamma (1)delta delta'chi psi rather than the expected gamma (2)tau (2)delta delta'chi psi. We observed the same heterogeneity when tau gamma mixed DnaX complexes were reconstituted in vitro. Re-examination of homomeric DnaX tau and gamma complexes assembled either in vitro or in vivo also revealed a stoichiometry of DnaX(3)delta delta'chi psi. Equilibrium sedimentation analysis showed that free DnaX is a tetramer in equilibrium with a free monomer. An assembly mechanism, in which the association of heterologous subunits with a homomeric complex alters the stoichiometry of the homomeric assembly, is without precedent. The significance of our findings to the architecture of the holoenzyme and the clamp-assembly apparatus of all other organisms is discussed.
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