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 ␤-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.

Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 °°°° C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ , a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor σ σ σ σ 32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °°°° C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ . Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ -independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.