Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis

Abstract

ATP-DnaA is the initiator of chromosomal replication inEscherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ϳ100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADPHda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA ؉ domain to be accessible to ATP bound to the DnaA AAA ؉ domain.The initiation of chromosomal replication is strictly regulated during the cell cycle. In Escherichia coli, a crucial target for this regulation is the formation of an active initiation complex, including the ATP-bound DnaA protein (ATP-DnaA) and the chromosomal replication origin, oriC (1, 2). The DiaA protein directly stimulates formation of this complex, which leads to the unwinding of duplex DNA within the oriC (3, 4). DnaB helicase then expands the unwound region to allow the loading of DnaG primase and DNA polymerase (pol) 5 III holoenzyme (5). The pol III holoenzyme consists of the pol III core, clamp (␤ subunit), and ␥ complex (clamp loader). The clamp forms a ring-shaped structure as a homodimer and is loaded onto the primed DNA by the ␥ complex to tether the pol III core onto DNA during DNA synthesis. After synthesis of the Okazaki fragments, the pol III core dissociates, and the sliding clamp remains on the DNA (5).There are at least three systems that repress extra initiations (2, 6, 7). First, binding of SeqA to hemimethylated oriC temporarily inhibits initiation complex formation (8 -10). The minimal oriC region contains 11 GATC sequence repeats that are available for modification by the DNA-adenine methyltransferase. Nascent strand synthesis transiently generates a hemimethylated oriC that is the target of SeqA. In the second system, there is a reduction of DnaA molecules that are accessible to the oriC. The datA locus is located near the oriC and ...

Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA

“…After replicational initiation, RIDA is activated by DNA loading of the pol III clamps, thereby promoting DnaA-ATP hydrolysis and repression of reinitiations. In fast growing cells, RIDA could be down-regulated in a cell cycle-coordinated manner by an unidentified pathway to assist the increase of the ATP-DnaA level (Kurokawa et al 1999;Su'etsugu et al 2008). Moreover, DnaA functions as a transcription repressor, and this activity is also stimulated by ATP binding (Messer and Weigel 1997;Gon et al 2006); e.g., ATP-DnaA represses dnaA transcription more tightly than ADP-DnaA (Speck et al 1999).…”

“…At the post-initiation stage, DnaA-ATP is hydrolyzed in a DNA-loaded b sliding clamp-dependent and ADP-Hda protein-dependent manner, yielding inactive ADP-DnaA (Katayama et al 1998;Kato and Katayama 2001;Su'etsugu et al 2005Su'etsugu et al , 2008. The b clamp is a subunit of the pol III holoenzyme, which is loaded onto DNA during DNA replication and remains attached to DNA after the creation of Okazaki fragments.…”

Regulating DNA Replication in Bacteria

“…4; Table 1) (Katayama et al 1998;Kato and Katayama 2001;Su'etsugu et al 2008). The resultant ADP-DnaA is inactive in initiation.…”

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA

“…In addition to this motif, Hda contains an AAAϩ domain with the Walker-type nucleotide-binding motif and several conserved amino acid sequence motifs (18,36). Hda AAAϩ does not bind ATP but specifically and stably binds to ADP to yield the activated monomeric form of Hda (19).…”