title: DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection.
abstract: Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known β-binding domain, decreasing interaction with the β2 processivity factor. Surprisingly, mutations within the β binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized.
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80303.
authors.forename: Janet C
authors.identifier: http://orcid.org/0000-0003-3235-3698
authors.initials: JC
authors.lastname: Lindow
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80303.
authors.forename: Paul R
authors.identifier: http://orcid.org/0000-0001-8461-9676
authors.initials: PR
authors.lastname: Dohrmann
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80303 charles.mchenry@colorado.edu.
authors.forename: Charles S
authors.identifier: http://orcid.org/0000-0001-6865-6119
authors.initials: CS
authors.lastname: McHenry
mesh_terms: D020816:Amino Acid Motifs; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D021122:Protein Subunits; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III
keywords: DNA polymerase; DNA polymerase III holoenzyme; DNA replication; Escherichia coli (E. coli); genetic screen; genetics; initiation complex formation; protein domain; protein-protein interactions; replicase