title: Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins.
abstract: Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30 degrees C. Whereas purified wild-type DnA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild-type protein. In a system reconstituted with purified proteins at 30 degrees C, the mutant protein initiated replication of single-stranded DNA that contains a DnA-binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA. Thermal stabilities of wild-type DnA and DnaAcos activities were comparable. Unlike wild-type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP-bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, California 94305, USA.
authors.forename: T
authors.initials: T
authors.lastname: Katayama
authors.forename: E
authors.initials: E
authors.lastname: Crooke
authors.forename: K
authors.initials: K
authors.lastname: Sekimizu
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria