An isolated Hda–clamp complex is functional in the regulatory inactivation of DnaA and DNA replication

• Published in: Journal of structural biology Vol. 156; no. 1; pp. 220 - 229
• Main Authors: Kawakami, Hironori, Su’etsugu, Masayuki, Katayama, Tsutomu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2006
• Subjects: AAA+; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - isolation & purification; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Clamp; DNA Polymerase III - metabolism; DNA Replication; DNA, Bacterial - metabolism; DnaA; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Feedback regulation; Hda; Histidine - chemistry; Holoenzymes - metabolism; Hydrolysis; Molecular Sequence Data; Mutation; Nucleotides - metabolism; Solubility; Feedback regulation; Clamp; DNA replication; DnaA; Hda; AAA+
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2006.02.007

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10–100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda–clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III ∗). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III ∗ and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.