title: The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase.
abstract: There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ'χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-C(tag)) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-C(tag) per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ'χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F' episome.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado-Boulder, 3415 Colorado Avenue, Boulder, CO 80303, USA.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Dohrmann
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Raul
authors.initials: R
authors.lastname: Correa
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Ryan L
authors.initials: RL
authors.lastname: Frisch
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA The Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Rosenberg
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado-Boulder, 3415 Colorado Avenue, Boulder, CO 80303, USA Charles.McHenry@colorado.edu.
authors.forename: Charles S
authors.initials: CS
authors.lastname: McHenry
mesh_terms: D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D018628:Gene Dosage; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D050296:Microbial Viability / Q000235:genetics; D009154:Mutation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D020035:Holoenzymes; D021122:Protein Subunits; C023140:DNA replicase; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase