Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins

Tsutomu Katayama, Elliott Crooke, Kazuhisa Sekimizu

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30°C. Whereas purified wild-type DnaA 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°C, the mutant protein initiated replication of single-stranded DNA that contains a DnaA-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 DnaA 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.

Original languageEnglish
Pages (from-to)813-820
Number of pages8
JournalMolecular Microbiology
Volume18
Issue number5
DOIs
Publication statusPublished - Dec 1995

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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