Protein Associations in DnaA-ATP Hydrolysis Mediated by the Hda-Replicase Clamp Complex

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 ␤-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.

Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis

“…In E. coli, the well characterized DnaA-related Hda protein regulates initiation by promoting the hydrolysis of DnaA-ATP to produce the inactive DnaA-ADP (3,29,30). In vitro, this hydrolysis requires Hda interaction with DnaA and with DnaN loaded onto the DNA and involves a conserved arginine-containing motif in the AAAϩ proteins (21,31). Whether in B. subtilis YabA also promotes a RIDA-like switch is not known.…”

“…In E. coli, the interactions with DnaA and DnaN are required for Hda-mediated RIDA (21). The DnaA-ATP hydrolysis in RIDA requires critical arginine residues that could cooperate in a catalytic center created by the interaction between the homologous AAAϩ domains of Hda and DnaA (21). However, in contrast to Hda, YabA shares no structural homology with DnaA, does not belong to the AAAϩ family of ATPases, and does not contain any DnaN-binding consensus motif.…”

“…In this context, the deletion of YabA, which abolished all of the interactions, does not suffice to establish the direct functional relationship between the interaction with DnaA and DnaN and initiation control. In E. coli, the interactions with DnaA and DnaN are required for Hda-mediated RIDA (21). The DnaA-ATP hydrolysis in RIDA requires critical arginine residues that could cooperate in a catalytic center created by the interaction between the homologous AAAϩ domains of Hda and DnaA (21).…”

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

“…Its modular architecture allows for interactions with different binding partners during the various steps of the formation of the active replication forks and coupling to other cellular events (3,15,17,20,22,29,30). In E. coli, DnaA III interactions with itself, Hda, or DnaC are important for the spatial positioning of the ATP-DnaA molecules and their recycling (13,17,31,32). Protein-protein interactions involving DnaA I-II play critical roles in DnaA self-oligomerization, helicase loading (7,10,17), and regulation of the replication timing (16,28).…”

Conservation of Eubacterial Replicases

“…The free b 2 sitting on this duplex DNA at the end of the newly synthesised fragment interacts indirectly with ATPDnaA through a bridge protein called Hda (18,19). Hda forms homodimers and two Hda homodimers can bind a single b 2 (20). The interactions to form DnaA-Hda 2 -b 2 promotes the conversion of ATP-DnaA to an inactive form (ADP-DnaA) which in turn inhibits inappropriate re-initiation at oriC during replication.…”