Chromosomal replication must be limited to once and only once per cell cycle. This is accomplished by multiple regulatory pathways that govern initiator proteins and replication origins. A principal feature of DNA replication is the coupling of the replication reaction to negative-feedback regulation. Some of the factors that are important in this process have been discovered, including the clamp (DNA polymerase III subunit-beta (DnaN)), the datA locus, SeqA, DnaA homologue protein (Hda) and YabA, as well as factors that are involved at other stages of the regulatory mechanism, such as DnaA initiator-associating protein (DiaA), the DnaA-reactivating sequence (DARS) loci and Soj. Here, we describe the regulation of DnaA, one of the central proteins involved in bacterial DNA replication, by these factors in Escherichia coli, Bacillus subtilis and Caulobacter crescentus.

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 Escherichia coli, the replication origin oriC consists of two functional regions: the duplex unwinding element (DUE) and its flanking DnaA-assembly region (DAR). ATP-DnaA molecules multimerize on DAR, unwinding DUE for DnaB helicase loading. However, DUE-unwinding mechanisms and functional structures in DnaA–oriC complexes supporting those remain unclear. Here, using various in vitro reconstituted systems, we identify functionally distinct DnaA sub-complexes formed on DAR and reveal novel mechanisms in DUE unwinding. The DUE-flanking left-half DAR carrying high-affinity DnaA box R1 and the ATP-DnaA-preferential DnaA box R5, τ1-2 and I1-2 sites formed a DnaA sub-complex competent in DUE unwinding and ssDUE binding, thereby supporting basal DnaB loading activity. This sub-complex is further subdivided into two; the DUE-distal DnaA sub-complex formed on the ATP–DnaA-preferential sites binds ssDUE. Notably, the DUE-flanking, DnaA box R1–DnaA sub-complex recruits DUE to the DUE-distal DnaA sub-complex in concert with a DNA-bending nucleoid protein IHF, thereby promoting DUE unwinding and binding of ssDUE. The right-half DAR–DnaA sub-complex stimulated DnaB loading, consistent with in vivo analyses. Similar features are seen in DUE unwinding of the hyperthermophile, Thermotoga maritima, indicating evolutional conservation of those mechanisms.

No abstract

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaAoriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific-oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.[Keywords: initiation of replication; protein complex; complex structure; DNA dynamics; cell cycle regulation; initiator regulation] Supplemental material is available at http://www.genesdev.org.