Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex.
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 beta-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. Arg(168) 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 approximately 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.
Su'etsugu
Masayuki
M
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Shimuta
Toh-Ru
TR
Ishida
Takuma
T
Kawakami
Hironori
H
Katayama
Tsutomu
T
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004265:DNA Helicases / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D046912:Multiprotein Complexes; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D015534:Trans-Activators; C045264:replication initiator protein; D000255:Adenosine Triphosphate; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; D000251:Adenosine Triphosphatases; C435211:Hda protein, E coli; D004265:DNA Helicases