Site-directed mutational analysis for the ATP binding of DnaA protein: Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo

Tohru Mizushima, Tohru Takaki, Toshio Kubota, Tomofusa Tsuchiya, Takeyoshi Miki, Tsutomu Katayama, Kazuhisa Sekimizu

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 → Ile or Asp-235 → Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.

Original languageEnglish
Pages (from-to)20847-20851
Number of pages5
JournalJournal of Biological Chemistry
Volume273
Issue number33
DOIs
Publication statusPublished - Aug 14 1998

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Carrier Proteins
Adenosine Triphosphate
Amino Acids
DNA Replication
Proteins
DNA
In Vitro Techniques
Protein Domains
Temperature
Adenosine Diphosphate
Escherichia coli
Assays
Defects
Mutation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Site-directed mutational analysis for the ATP binding of DnaA protein : Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo. / Mizushima, Tohru; Takaki, Tohru; Kubota, Toshio; Tsuchiya, Tomofusa; Miki, Takeyoshi; Katayama, Tsutomu; Sekimizu, Kazuhisa.

In: Journal of Biological Chemistry, Vol. 273, No. 33, 14.08.1998, p. 20847-20851.

Research output: Contribution to journalArticle

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AU - Miki, Takeyoshi

AU - Katayama, Tsutomu

AU - Sekimizu, Kazuhisa

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