DNA polymerase III holoenzyme of Escherichia coli. I. Purification and distinctive functions of subunits tau and gamma, the dnaZX gene products.
Escherichia coli dnaZX, the gene which when mutant blocks DNA chain elongation, was cloned into a lambda PL promoter-mediated expression vector. In cells carrying this plasmid, the activity that complements a mutant dnaZ extract in replicating a primed single-stranded DNA circle was increased about 20-fold. Two polypeptides of 71 and 52 kDa were overproduced. Upon fractionation, two complementing activities were purified to homogeneity and proved to be the 71- and 52-kDa polypeptides. Immunoassays revealed their respective identities with the tau and gamma subunits of DNA polymerase III holoenzyme. The N-terminal amino acid sequences of the first 12 residues were identical in both subunits, as were their molar specific activities in dnaZ complementation. Thus, the tau subunit complements the defect in the mutant holoenzyme from the dnaZts strain as efficiently as does the gamma subunit. Inasmuch as the 71-kDa subunit (tau) can also overcome the enzymatic defect in a dnaX mutant strain, this polypeptide has dual replication functions, only one of which can be performed by the gamma subunit. Availability of pure tau and gamma subunits for study has provided the basis for proposing an asymmetry in the structure and function of a dimeric DNA polymerase III holoenzyme.
Maki
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Department of Biochemistry, Stanford University School of Medicine, California 94305.
Kornberg
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D003001:Cloning, Molecular; D004258:DNA Polymerase III / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic
D046911:Macromolecular Substances; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase