title: The A184V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermolability in initiation of Escherichia coli DNA replication.
abstract: The temperature-sensitive dnaA5 and dnaA46 alleles each contain two missense mutations. These mutations have been separated and the resulting mutant proteins studied with regard to their role in initiation of DNA replication in vitro. Whereas the His-252 to tyrosine substitution (H252Y) unique to the dnaA46 allele did not affect the activities of DnaA protein, the unique substitution of the dnaA5 allele, Gly-426 to serine (G426S), was reduced in its DNA-binding affinity for oriC, the chromosomal origin. This suggests that the C-terminal region of the DnaA protein is involved in DNA binding. The alanine-to-valine substitution at amino acid 184 (A184V) that is common to both of the alleles is responsible for the thermolabile defect and lag in DNA synthesis of these mutants. Mutant proteins bearing the common substitution were defective in ATP binding and were inactive in a replication system reconstituted with purified proteins. DnaK and GrpE protein activated these mutant proteins for replication and ATP binding; the latter was measured indirectly by the ATP-dependent formation of a trypsin-resistant peptide. However, with this assay, the ATP-binding affinity appeared to be reduced relative to wild-type DnaA protein. Activation was by conversion of a self-aggregate to the monomer, and also by a conformational alteration that correlated with ATP binding.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824-1319, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Carr
authors.forename: J M
authors.initials: JM
authors.lastname: Kaguni
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D016296:Mutagenesis; D017354:Point Mutation; D018741:Replication Origin; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli