Escherichia coli DnaA protein, a member of the AAA؉ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaAspecific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATPDnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the Arg-285 residue for initiation. Sequence and structural analyses suggest that the DnaA Arg-285 residue is an arginine finger, an AAA؉ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA Arg-285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaAspecific binding sites in the complex.DnaA protein plays an essential role in the initiation of Escherichia coli chromosomal replication (1-4). The protein forms a homomultimer on the replication origin oriC to form an initiation complex. When the ATP-bound form of DnaA is included in the initiation complex, a region within oriC containing AT-rich 13-mers is specifically unwound, resulting in open complex formation. ADP-DnaA forms a multimeric complex on oriC that does not promote open complex formation. On the exposed single-stranded (ss) 1 DNA of the 13-mer region, DnaB helicase is loaded via ordered interactions with the DnaC helicase loader and DnaA. DnaG primase forms a complex with DnaB that promotes DNA duplex unwinding and RNA primer synthesis, which allow loading of the DNA polymerase (pol) III holoenzyme. The pol III holoenzyme consists of the  clamp subunit, which is directly loaded onto the primed site, and the pol III* subassembly, which binds to the  clamp. Pol III* includes the clamp-loader ␥ complex and the pol III core complex, which contains the catalytic center of the polymerase.
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 ...
In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a pre-Replicative Complex (pre-RC) with a MCM2-7 double-hexamer encircling DNA. Using purified components in the presence of ATPγS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the hetero-heptameric ORC-Cdc6 and the hetero-heptameric Cdt1-MCM2-7, called the OCCM complex. Cryo-EM studies of the 14-protein complex reveal that the two separate heptameric complexes are extensively engaged, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. ORC-Cdc6 undergoes a concerted conformational change into a right-handed spiral with the helical symmetry identical to the DNA double helix. The results show a striking structural similarity between the ORC-Cdc6 helicase loader and the Replication Factor-C clamp loader and suggest a conserved mechanism of action.
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.
This review summarizes the mechanisms of the initiator protein DnaA in replication initiation and its regulation in Escherichia coli. The chromosomal origin (oriC) DNA is unwound by the replication initiation complex to allow loading of DnaB helicases and replisome formation. The initiation complex consists of the DnaA protein, DnaA-initiator-associating protein DiaA, integration host factor (IHF), and oriC, which contains a duplex-unwinding element (DUE) and a DnaA-oligomerization region (DOR) containing DnaA-binding sites (DnaA boxes) and a single IHF-binding site that induces sharp DNA bending. DiaA binds to DnaA and stimulates DnaA assembly at the DOR. DnaA binds tightly to ATP and ADP. ATP-DnaA constructs functionally different sub-complexes at DOR, and the DUE-proximal DnaA sub-complex contains IHF and promotes DUE unwinding. The first part of this review presents the structures and mechanisms of oriC-DnaA complexes involved in the regulation of replication initiation. During the cell cycle, the level of ATP-DnaA level, the active form for initiation, is strictly regulated by multiple systems, resulting in timely replication initiation. After initiation, regulatory inactivation of DnaA (RIDA) intervenes to reduce ATP-DnaA level by hydrolyzing the DnaA-bound ATP to ADP to yield ADP-DnaA, the inactive form. RIDA involves the binding of the DNA polymerase clamp on newly synthesized DNA to the DnaA-inactivator Hda protein. In datA-dependent DnaA-ATP hydrolysis (DDAH), binding of IHF at the chromosomal locus datA, which contains a cluster of DnaA boxes, results in further hydrolysis of DnaA-bound ATP. SeqA protein inhibits untimely initiation at oriC by binding to newly synthesized oriC DNA and represses dnaA transcription in a cell cycle dependent manner. To reinitiate DNA replication, ADP-DnaA forms oligomers at DnaA-reactivating sequences (DARS1 and DARS2), resulting in the dissociation of ADP and the release of nucleotide-free apo-DnaA, which then binds ATP to regenerate ATP-DnaA. In vivo, DARS2 plays an important role in this process and its activation is regulated by timely binding of IHF to DARS2 in the cell cycle. Chromosomal locations of DARS sites are optimized for the strict regulation for timely replication initiation. The last part of this review describes how DDAH and DARS regulate DnaA activity.
The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here we report single particle cryo-EM-derived structure of the supra-molecular assembly comprising of S. cerevisiae ORC, the replication initiation factor Cdc6 and double strand ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3 with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, particularly re-orientating the Orc1 N-terminal BAH-domain. Segmentation of the 3D map of ORC•Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.
In Escherichia coli, the level of the ATP–DnaA initiator is increased temporarily at the time of replication initiation. The replication origin, oriC, contains a duplex-unwinding element (DUE) flanking a DnaA-oligomerization region (DOR), which includes twelve DnaA-binding sites (DnaA boxes) and the DNA-bending protein IHF-binding site (IBS). Although complexes of IHF and ATP–DnaA assembly on the DOR unwind the DUE, the configuration of the crucial nucleoprotein complexes remains elusive. To resolve this, we analyzed individual DnaA protomers in the complex and here demonstrate that the DUE–DnaA-box-R1–IBS–DnaA-box-R5M region is essential for DUE unwinding. R5M-bound ATP–DnaA predominantly promotes ATP–DnaA assembly on the DUE-proximal DOR, and R1-bound DnaA has a supporting role. This mechanism might support timely assembly of ATP–DnaA on oriC. DnaA protomers bound to R1 and R5M directly bind to the unwound DUE strand, which is crucial in replication initiation. Data from in vivo experiments support these results. We propose that the DnaA assembly on the IHF-bent DOR directly binds to the unwound DUE strand, and timely formation of this ternary complex regulates replication initiation. Structural features of oriC support the idea that these mechanisms for DUE unwinding are fundamentally conserved in various bacterial species including pathogens.
Background: ATP-DnaA molecules oligomerize and form two subcomplexes on the replication origin. Results: The Arg fingers of DnaA bound at the outer edges of the DnaA complexes are oriented inward within the origin. Conclusion: The Arg fingers, but not bound ATP, of the outer edge DnaA protomers promote construction of active initiation complexes. Significance: An important mechanical basis in the initiation complex is revealed.
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