title: A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader.
abstract: The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.
authors.affiliation: Department of Chemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
authors.forename: B
authors.initials: B
authors.lastname: Ason
authors.forename: J G
authors.initials: JG
authors.lastname: Bertram
authors.forename: M M
authors.initials: MM
authors.lastname: Hingorani
authors.forename: J M
authors.initials: JM
authors.lastname: Beechem
authors.forename: M
authors.initials: M
authors.lastname: O'Donnell
authors.forename: M F
authors.initials: MF
authors.lastname: Goodman
authors.forename: L B
authors.initials: LB
authors.lastname: Bloom
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D004258:DNA Polymerase III / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D013379:Substrate Specificity; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004258:DNA Polymerase III