Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex

Masayuki Su'etsugu, Toh Ru Shimuta, Takuma Ishida, Hironori Kawakami, Tsutomu Katayama

Research output: Contribution to journalArticle

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Abstract

In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the β-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg168 in the AAA+ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain ∼50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA+ proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA+ domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

Original languageEnglish
Pages (from-to)6528-6536
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number8
DOIs
Publication statusPublished - Feb 25 2005

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Clamping devices
Hydrolysis
Adenosine Triphosphate
Proteins
Dimers
Holoenzymes
Mutant Proteins
Adenosine Diphosphate
Escherichia coli
Assays
Molecules
DNA
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex. / Su'etsugu, Masayuki; Shimuta, Toh Ru; Ishida, Takuma; Kawakami, Hironori; Katayama, Tsutomu.

In: Journal of Biological Chemistry, Vol. 280, No. 8, 25.02.2005, p. 6528-6536.

Research output: Contribution to journalArticle

Su'etsugu, Masayuki ; Shimuta, Toh Ru ; Ishida, Takuma ; Kawakami, Hironori ; Katayama, Tsutomu. / Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 8. pp. 6528-6536.
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