Hironori Kawakami

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.

Protein Associations in DnaA-ATP Hydrolysis Mediated by the Hda-Replicase Clamp Complex

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 β-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. Arg168 in the AAA+ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain ∼50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA+ proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA+ domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC–Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC–Cdc6 and Cdt1–MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC–Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC–Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC–Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.

Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

DnaA, ORC, and Cdc6: Similarity beyond the domains of life and diversity

To initiate chromosomal DNA replication, specific proteins bind to the replication origin region and form multimeric and dynamic complexes. Bacterial DnaA, the eukaryotic origin recognition complex (ORC), and Cdc6 proteins, most of which include an AAA+(-like) motif, play crucial roles in replication initiation. The importance of ATP binding and hydrolysis in these proteins has recently become recognized. ATP binding of Escherichia coli DnaA is required for the formation of the activated form of a DnaA multimer on the replication origin. The ATP-DnaA multimer can unwind duplex DNA in an origin-dependent manner, which is supported by various specific functions of several AAA+ motifs. DnaA-ATP hydrolysis is stimulated after initiation, repressing extra initiations, and sustaining once-per-cell cycle replication. ATP binding of ORC and Cdc6 in Saccharomyces cerevisiae is required for heteromultimeric complex formation and specific DNA binding. ATP hydrolysis of these proteins is important for the efficient loading of the minichromosome maintenance protein complex, a component of the putative replicative helicase. In this review, we discuss the roles of DnaA, ORC, and Cdc6 in replication initiation and its regulation. We also summarize the functional features of the AAA+ domains of these proteins, and the functional divergence of ORC in chromosomal dynamics.

DNA replication-coupled inactivation of DnaA protein in vitro: a role for DnaA arginine-334 of the AAA+ Box VIII motif in ATP hydrolysis.

The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication‐stimulated hydrolysis of DnaA‐bound ATP yields the ADP‐bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7‐fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA‐ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild‐type DnaA is hydrolysed in a DNA‐dependent intrinsic manner or in a sliding clamp‐dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.

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.

Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA

Abstract. In Escherichia coli, an interaction between the replication initiator DnaA and the sliding clamp protein, the β subunit (DnaN) of DNA polymerase III, is required to regulate the chromosomal replication cycle. We report here that colony formation by, and cell division of, the temperature (42°C)-sensitive dnaN59 mutant are inhibited at 34–35°C when DnaA is moderately (4-to 8-fold ) overexpressed, although chromosomal replication and the β subunit-dependent regulation of DnaA activity are not significantly inhibited. Immunoblotting analysis revealed that the β subunit is abundant (present at a level of about 5000 dimers per cell) at 34°C, and its concentration per unit cell volume was practically unaffected in the dnaN59 mutant by the overexpression of DnaA. The dnaN mutant cells that overexpress DnaA become filamentous at 34°C via an sfiA-independent pathway, different from that activated by the SOS response. This filamentation is accompanied by inhibition of nucleoid partition and FtsZ ring formation. In the dnaN59 mutant, oversupply of DnaA may disturb the coordinated action of cell cycle-regulating molecules, thus leading to the inhibition of these events.

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.

The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

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. ATP-DnaA binds to multiple DnaA boxes in the Escherichia coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg-285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that had alternative DNA binding sequence specificity and chimeric oriC molecules bearing the alternative DnaA binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg-285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg-285 fingers face inward to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases.