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A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of δδ′ with DnaX 4 forms DnaX 3 δδ′
Author(s) -
Pritchard Arthur E.,
Dallmann H.Garry,
Glover Bradley P.,
McHenry Charles S.
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.23.6536
Subject(s) - homomeric , operon , biology , tetramer , polymerase , biophysics , dna polymerase , heterologous , microbiology and biotechnology , dna , protein subunit , biochemistry , escherichia coli , enzyme , gene
We have constructed a plasmid‐borne artificial operon that expresses the six subunits of the DnaX complex of Escherichia coli DNA polymerase III holoenzyme: τ, γ, δ, δ′, χ and ψ. Induction of this operon followed by assembly in vivo produced two τγ mixed DnaX complexes with stoichiometries of τ 1 γ 2 δδ′χψ and τ 2 γ 1 δδ′χψ rather than the expected γ 2 τ 2 δδ′χψ. We observed the same heterogeneity when τγ mixed DnaX complexes were reconstituted in vitro . Re‐examination of homomeric DnaX τ and γ complexes assembled either in vitro or in vivo also revealed a stoichiometry of DnaX 3 δδ′χψ. 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.