Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation

Control of DNA replication initiation is essential for bacterial cells to co-ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP-dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.     

The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation

DNA replication and chromosome segregation must be carefully regulated to ensure reproductive success. During Bacillus subtilis sporulation, chromosome copy number is reduced to two, and cells divide asymmetrically to produce the future spore (forespore) compartment. For successful sporulation, oriC must be captured in the forespore. New rounds of DNA replication are prevented in part by SirA, a protein that utilizes residues in its N‐terminus to directly target Domain I of the bacterial initiator, DnaA. Using a quantitative forespore chromosome organization assay, we show that SirA also acts in the same pathway as another DnaA regulator, Soj, to promote oriC capture in the forespore. By analyzing loss‐of‐function variants of both SirA and DnaA, we observe that SirA's ability to inhibit DNA replication can be genetically separated from its role in oriC capture. In addition, we identify substitutions near the C‐terminus of SirA and in DnaA Domain III that enhance interaction between the two proteins. One such variant, SirA_P141T, remained functional in regard to inhibiting replication, but was unable to support oriC capture. Collectively, our results support a model in which SirA targets DnaA Domain I to inhibit DNA replication, and DnaA Domain III to facilitate Soj‐dependent oriC capture in the forespore.     

Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication

DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~ 15 Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication.     

Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA

Control of DNA replication initiation is essential for normal cell growth. A unifying characteristic of DNA replication initiator proteins across the kingdoms of life is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains, that in vitro stretches DNA to promote replication origin opening. The Bacillus subtilis protein Soj/ParA has previously been shown to regulate DnaA-dependent DNA replication initiation; however, the mechanism underlying this control was unknown. Here, we report that Soj directly interacts with the AAA+ domain of DnaA and specifically regulates DnaA helix assembly. We also provide critical biochemical evidence indicating that DnaA assembles into a helical oligomer in vivo and that the frequency of replication initiation correlates with the extent of DnaA oligomer formation. This work defines a significant new regulatory mechanism for the control of DNA replication initiation in bacteria.     

Characterization of the Soj/Spo0J chromosome segregation proteins and identification of putative parS sequences in Helicobacter pylori

The Soj and Spo0J proteins, together with one or more parS sequences, are crucial to chromosome segregation and the progression of cell cycle in many bacteria. In Helicobacter pylori, genes coding for Soj and a plasmid replication-partition-related protein containing a Spo0J or ParB conserved domain, together with two putative parS sites identified in this study, were found to be located within the origin-proximal 20-30% of the circular chromosome. Recombinant H. pylori Spo0J bound specifically to the two putative parS sequences and that of Bacillus subtilis. In addition, hydrolysis of ATP by H. pylori Soj was accelerated in the presence of parS and/or Spo0J. Protein-protein interactions, intracellular levels, and subcellular localization of Soj and Spo0J were analyzed through polyclonal antibodies directed against recombinant Soj and Spo0J. This study was the first implication of the existence of a functional parABS system in H. pylori.     

Participation of chromosome segregation protein ParAI of Vibrio cholerae in chromosome replication

Vibrio cholerae carries homologs of plasmid-borne parA and parB genes on both of its chromosomes. The par genes help to segregate many plasmids and chromosomes. Here we have studied the par genes of V. cholerae chromosome I. Earlier studies suggested that ParBI binds to the centromeric site parSI near the origin of replication (oriI), and parSI-ParBI complexes are placed at the cell poles by ParAI. Deletion of parAI and parSI caused the origin-proximal DNA to be less polar. Here we found that deletion of parBI also resulted in a less polar localization of oriI. However, unlike the deletion of parAI, the deletion of parBI increased the oriI number. Replication was normal when both parAI and parBI were deleted, suggesting that ParBI mediates its action through ParAI. Overexpression of ParAI in a parABI-deleted strain also increased the DNA content. The results are similar to those found for Bacillus subtilis, where ParA (Soj) stimulates replication and this activity is repressed by ParB (SpoOJ). As in B. subtilis, the stimulation of replication most likely involves the replication initiator DnaA. Our results indicate that control of chromosomal DNA replication is an additional function of chromosomal par genes conserved across the Gram-positive/Gram-negative divide.     

YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA

Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins is their distinctive AAA+ nucleotide‐binding domains. The bacterial initiator DnaA assembles into a right‐handed helical oligomer built upon interactions between neighbouring AAA+ domains to form an active initiation complex. Recently we developed a unique cross‐linking assay that specifically detects ATP‐dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Escherichia coli DnaA where ATP binding appears to be the targeted activity.     

Control of the replication initiator DnaA by an anti-cooperativity factor

Proper coordination of DNA replication with cell growth and division is critical for production of viable progeny. In bacteria, coordination of DNA replication with cell growth is generally achieved by controlling activity of the replication initiator DnaA and its access to the chromosomal origin of replication, oriC. Here we describe a previously unknown mechanism for regulation of DnaA. YabA, a negative regulator of replication initiation in Bacillus subtilis, interacts with DnaA and DnaN, the sliding (processivity) clamp of DNA polymerase. We found that in vivo, YabA associated with the oriC region in a DnaA-dependent manner and limited the amount of DnaA at oriC. In vitro, purified YabA altered binding of DnaA to DNA by inhibiting cooperativity. Although previously undescribed, proteins that directly inhibit cooperativity may be a common mechanism for regulating replication initiation. Conditions that cause release of DnaN from the replisome, or overproduction of DnaN, caused decreased association of YabA and increased association of DnaA with oriC. This effect of DnaN, either directly or indirectly, is likely responsible, in part, for enabling initiation of a new round of replication following completion of a previous round.     

Site-directed mutational analysis of DnaA protein, the initiator of chromosomal DNA replication in E. coli

DnaA protein, the initiator for chromosomal DNA replication in Escherichia coli, has various activities, such as oligomerization (DnaA-DnaA interaction), ATP-binding, ATPase activity and membrane-binding. Site-directed mutational analyses have revealed not only the amino acid residues that are essential for these activities but also the functions of these activities. Following is a summary of the functions and regulatory mechanisms of DnaA protein in the initiation of chromosomal DNA replication. ATP-bound DnaA protein, but not other forms of the protein binds to the origin of DNA replication and forms oligomers to open-up the duplex DNA. This oligomerization is mediated by a DnaA-DnaA interaction through the N-terminal region of the protein. After initiation of DNA replication, the ATPase activity of DnaA protein is stimulated and DnaA protein is inactivated to the ADP-bound form to suppress the re-initiation of DNA replication. DnaA protein binds to acidic phospholipids through an ionic interaction between basic amino acid residues of the protein and acidic residues of phospholipids. This interaction seems to be involved in the re-activation of DnaA protein (from the ADP-bound form to the ATP-bound form) to initiate DNA replication after the appropriate interval.     

Control of the bacterial cell cycle

New insights into the control of DNA replication through growth, hemimethylated DNA and DnaA protein have been described. Fundamental shifts in thinking have resulted in the identification of new cell cycle genes with potential roles in initiation of DNA replication, chromosomal segregation and division. Excitingly, this trend may also narrow the apparent differences between the prokaryotic and eukaryotic cell cycles.     

The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC

Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram-negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaA(TB)) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli, ATPase activity of DnaA(TB) promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaA(TB) proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC-DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaA(TB) bound to DnaA boxes similarly with ATP or ADP. DnaA(TB) binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation.