A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX(4) forms DnaX(3)deltadelta'.
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 taugamma mixed DnaX complexes with stoichiometries of tau(1)gamma(2)deltadelta'chipsi and tau(2)gamma(1)deltadelta'chipsi rather than the expected gamma(2)tau(2)deltadelta'chipsi. We observed the same heterogeneity when taugamma 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)deltadelta'chipsi. 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.
Pritchard
A E
AE
Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Dallmann
H G
HG
Glover
B P
BP
McHenry
C S
CS
D000645:Ammonium Sulfate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D002847:Chromatography, Agarose; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D020035:Holoenzymes / Q000737:chemistry*; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D014461:Ultracentrifugation
D001426:Bacterial Proteins; C103076:DnaX protein, Bacteria; D020035:Holoenzymes; D004258:DNA Polymerase III; D000645:Ammonium Sulfate