Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372

Masaki Makise, Shinji Mima, Motohiro Koterasawa, Tomofusa Tsuchiya, Tohru Mizushima

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. Acidic phospholipids decrease its affinity for adenine nucleotides, and re-activate the ADP-bound form to the ATP-bound form. We have previously reported that two mutant forms, DnaAR328E and DnaAK372E, have decreased affinity for cardiolipin (CL). In the present study, we constructed a mutant DnaA protein, DnaA435, with both R328E and K372E, and compared its biochemical characteristics with those of DnaAR328E and DnaAK372E. DnaA435 could bind to oriC DNA, but did not bind ATP or ADP. In DnaA435, compared with DnaAR328E and DnaAK372E, CL caused less inhibition of oriC DNA binding, suggesting that amino acids R328 and K372 are involved in the interaction of DnaA with acidic phospholipids. DnaA435 could initiate DNA synthesis on oriC both in vivo and in vitro. Based on these results, we propose that ATP activates DnaA protein by changing its higher order structure around R328 and K372.

Original languageEnglish
Pages (from-to)453-458
Number of pages6
JournalBiochemical Journal
Volume362
Issue number2
DOIs
Publication statusPublished - 2002 Mar 1
Externally publishedYes

Fingerprint

Cardiolipins
Adenosine Triphosphate
Adenosine Diphosphate
Mutation
DNA
Phospholipids
Proteins
Adenine Nucleotides
Mutant Proteins
DNA Replication
Escherichia coli
Amino Acids
In Vitro Techniques

Keywords

  • Acidic phospholipids
  • ATP binding
  • Escherichia coli
  • Mutational analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Makise, M., Mima, S., Koterasawa, M., Tsuchiya, T., & Mizushima, T. (2002). Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372 Biochemical Journal, 362(2), 453-458. https://doi.org/10.1042/0264-6021:3620453

Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372 . / Makise, Masaki; Mima, Shinji; Koterasawa, Motohiro; Tsuchiya, Tomofusa; Mizushima, Tohru.

In: Biochemical Journal, Vol. 362, No. 2, 01.03.2002, p. 453-458.

Research output: Contribution to journalArticle

Makise, M, Mima, S, Koterasawa, M, Tsuchiya, T & Mizushima, T 2002, 'Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372 ', Biochemical Journal, vol. 362, no. 2, pp. 453-458. https://doi.org/10.1042/0264-6021:3620453
Makise, Masaki ; Mima, Shinji ; Koterasawa, Motohiro ; Tsuchiya, Tomofusa ; Mizushima, Tohru. / Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372 In: Biochemical Journal. 2002 ; Vol. 362, No. 2. pp. 453-458.
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