title: Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading.
abstract: The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Magdalena M
authors.initials: MM
authors.lastname: Felczak
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Jay M
authors.initials: JM
authors.lastname: Sage
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Hupert-Kocurek
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Senem
authors.initials: S
authors.lastname: Aykul
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319 kaguni@msu.edu.
authors.forename: Jon M
authors.initials: JM
authors.lastname: Kaguni
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053488:DnaB Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature / Q000009:adverse effects; D007700:Kinetics; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018741:Replication Origin
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C506730:dnaB protein, E coli; D004265:DNA Helicases; D053488:DnaB Helicases
keywords: DNA replication; DNA-protein interaction; Escherichia coli (E. coli); bacterial genetics; nucleic acid enzymology