Shogo Ozaki

Regulation of the replication cycle: conserved and diverse regulatory systems for DnaA and oriC

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-β (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.

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

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). ATP-DnaA 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 ATP-specific 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, ATP-DnaA-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.

DnaA structure, function, and dynamics in the initiation at the chromosomal origin

Escherichia coli DnaA is the initiator of chromosomal replication. Multiple ATP-DnaA molecules assemble at the oriC replication origin in a highly regulated manner, and the resultant initiation complexes promote local duplex unwinding within oriC, resulting in open complexes. DnaB helicase is loaded onto the unwound single-stranded region within oriC via interaction with the DnaA multimers. The tertiary structure of the functional domains of DnaA has been determined and several crucial residues in the initiation process, as well as their unique functions, have been identified. These include specific DNA binding, inter-DnaA interaction, specific and regulatory interactions with ATP and with the unwound single-stranded oriC DNA, and functional interaction with DnaB helicase. An overall structure of the initiation complex is also proposed. These are important for deepening our understanding of the molecular mechanisms that underlie DnaA assembly, oriC duplex unwinding, regulation of the initiation reaction, and DnaB helicase loading. In this review, we summarize recent progress on the molecular mechanisms of the functions of DnaA on oriC. In addition, some members of the AAA+ protein family related to the initiation of replication and its regulation (e.g., DnaA) are briefly discussed.

Highly organized DnaA-oriC complexes recruit the single-stranded DNA for replication initiation.

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.

The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif

Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP‐binding‐dependent 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.

Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex

Background: Multiple DnaA molecules form highly ordered complexes on the origin DNA to initiate chromosomal replication. Results: Novel structural motifs of DnaA are specifically required for the formation of the DNA unwinding-specific DnaA subcomplex. Conclusion: Distinct inter-DnaA interactions are required for the unwinding-specific subcomplex. Significance: Differentiation of the unwinding-specific subcomplex and a key mechanism underlying it are revealed. In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.

The DnaA homolog of the hyperthermophilic eubacterium Thermotoga maritima forms an open complex with a minimal 149-bp origin region in an ATP-dependent manner

In Escherichia coli, ATP‐DnaA, but not ADP‐DnaA, forms an initiation complex that undergoes site‐specific duplex DNA unwinding, open complex formation. However, it remains unclear how highly the ATP‐dependent activation of the initiation factor is conserved in evolution. The hyperthermophile Thermotoga maritima is one of the most ancient eubacteria in evolution. Here, we show that the DnaA homolog (tmaDnaA) of this bacterium forms open complexes with the predicted origin region (tma‐oriC) in vitro. TmaDnaA has a strong and specific affinity for ATP/ADP as well as for 12‐mer repeating sequences within the tma‐oriC. Unlike ADP‐tmaDnaA, ATP‐tmaDnaA is highly cooperative in DNA binding and forms open complexes in a manner that depends on temperature and the superhelical tension of the tma‐oriC‐bearing plasmid. The minimal tma‐oriC required for unwinding is a 149‐bp region containing five repeats of the 12‐mer sequence and two AT‐rich 9‐mer repeats. TmaDnaA‐binding to the 12‐mer motif provokes DNA bending. The 9‐mer region is the duplex‐unwinding site. The tmaDnaA‐binding and unwinding motifs of tma‐oriC share sequence homology with corresponding archaeal and eukaryotic sequences. These findings suggest that the ATP‐dependent molecular switch of the initiator and the mechanisms in the replication initiation complex are highly conserved in eubacterial evolution.

A Replicase Clamp-Binding Dynamin-like Protein Promotes Colocalization of Nascent DNA Strands and Equipartitioning of Chromosomes in E. coli

In Escherichia coli, bidirectional chromosomal replication is accompanied by the colocalization of sister replication forks. However, the biological significance of this mechanism and the key factors involved are still largely unknown. In this study, we found that a protein, termed CrfC, helps sustain the colocalization of nascent DNA regions of sister replisomes and promote chromosome equipartitioning. CrfC formed homomultimers that bound to multiple molecules of the clamp, a replisome subunit that encircles DNA, and colocalized with nascent DNA regions in a clamp-binding-dependent manner in living cells. CrfC is a dynamin homolog; however, it lacks the typical membrane-binding moiety and instead possesses a clamp-binding motif. Given that clamps remain bound to DNA after Okazaki fragment synthesis, we suggest that CrfC sustains the colocalization of sister replication forks in a unique manner by linking together the clamp-loaded nascent DNA strands, thereby laying the basis for subsequent chromosome equipartitioning.

Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli

Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature‐sensitive dnaA508 and dnaA167 mutants at 40 °C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ, a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor σ32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ. Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ‐independent manner. Gel‐filtration and chemical cross‐linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat‐shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.

Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif

In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37 °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37°C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.