Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATPDnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATPspecific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATPDnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. In Escherichia coli, DnaA forms a stable complex with ATP or ADP and binds to 9-mer sequences called DnaA boxes within the replication origin oriC, resulting in the formation of homomultimeric complexes (1-4). A DnaA-binding protein, DiaA, directly stimulates formation of ATP-DnaA multimers on oriC (5, 6). ATP-DnaA multimers, but not ADP-DnaA multimers, promote specific inter-DnaA interactions on oriC, resulting in the adoption of an activated conformation as the initiation complexes, which interact with ATP-DnaA-specific low affinity sites within oriC (7-9). This conformational change triggers duplex unwinding of the ATrich 13-mer repeats (DNA unwinding element (DUE) 4 ) within oriC with the aid of the superhelicity of DNA and heat energy, creating open complexes (10, 11). The mechanisms and functional structures within DnaA directly responsible for the ATPDnaA-specific duplex unwinding remain unexplored.Open complex formation is a critical regulatory point for determining whether replicational initiation will occur during the cell cycle (1, 2). DnaB helicase is loaded onto the singlestranded (ss) region in open complexes in a manner depending on a DnaA-DnaB interaction and the DnaC helicase loader. The loaded helicase expands the ssDNA region, which leads to the assembly of replication machineries, including DnaG primase and ...

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SummaryEscherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-bindingdependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10-to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10-to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.

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 ...