Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo

The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature‐sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI‐mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes.     

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.     

The Bacillus subtilis DnaD and DnaB proteins exhibit different DNA remodelling activities

Primosomal protein cascades load the replicative helicase onto DNA. In Bacillus subtilis a putative primosomal cascade involving the DnaD-DnaB-DnaI proteins has been suggested to participate in both the DnaA and PriA-dependent loading of the replicative helicase DnaC onto the DNA. Recently we discovered that DnaD has a global remodelling DNA activity suggesting a more widespread role in bacterial nucleoid architecture. Here, we show that DnaB forms a "square-like" tetramer with a hole in the centre and suggest a model for its interaction with DNA. It has a global DNA remodelling activity that is different from that of DnaD. Whereas DnaD opens up supercoiled DNA, DnaB acts as a lateral compaction protein. The two competing activities can act together on a supercoiled plasmid forming two topologically distinct poles; one compacted with DnaB and the other open with DnaD. We propose that the primary roles of DnaB and DnaD are in bacterial nucleoid architecture control and modulation, and their effects on the initiation of DNA replication are a secondary role resulting from architectural perturbations of chromosomal DNA.     

Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication

Initiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB-coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.     

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication,SirA, with domain I of DnaA

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA‐interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N‐terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α‐helical with predominantly apolar side‐chains packing in a hydrophobic interface. Site‐directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA‐interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.     

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.     

DnaD protein of Bacillus subtilis interacts with DnaA, the initiator protein of replication

The yeast two-hybrid assay revealed that Bacillus subtilis DnaD, a possible component of the primosome and required for replication initiation, interacted with DnaA and DnaD itself. The mutant DnaD23 was incapable of interacting with DnaA but retained interaction with the wild-type DnaD. These results suggest that interaction between DnaD and DnaA is important for replication initiation.     

Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis

The Bacillus subtilis proteins DnaD and DnaB are essential for replication initiation and are conserved in low G+C content Gram-positive bacteria. Previous work indicated that DnaD and DnaB are involved in helicase loading during the process of restarting stalled replication forks. We have investigated the roles of DnaD and DnaB in replication initiation at oriC in vivo. Using chromatin immunoprecipitation (ChIP), we found that DnaD and DnaB functions are needed to load the replicative helicase at oriC. To investigate further the functions of DnaD and DnaB in replication initiation, we isolated and characterized suppressors of the temperature sensitivity of dnaD and dnaB mutant cells. In both cases, we isolated the identical missense mutation in dnaB, dnaBS371P. Using yeast two-hybrid analysis, we found that dnaBS371P uncovers a previously undetected physical interaction between DnaD and DnaB. We also found that DnaBS371P constitutively recruits DnaD to the membrane fraction of cells, where DnaB and oriC are enriched. Phenotypes of cells expressing DnaBS371P are consistent with aberrant replication control. We hypothesize that B. subtilis regulates replication initiation by regulating a physical interaction between two proteins essential for helicase loading at chromosomal origins.     

Characterization of physical interaction between replication initiator protein DnaA and replicative helicase from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> H37Rv

In the pathogenic Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, the genetic and biochemical mechanisms for initiation of DNA replication are largely unknown. In the present study, we have characterized the physical interactions between M. tuberculosis DnaA and DnaB using both in vivo methods, such as bacterial two-hybrid assays, and in vitro techniques, such as surface plasmon resonance (SPR) and Pull-down/Western blotting. The full-length N-terminus (1–206 residues) of DnaB was found to interact with DnaA, while the shorter N-terminal domain of DnaB (1–125 residues), which lacked the linker region, did not. Further SPR and electrophoretic mobility shift assays indicated that the N-terminus (1–206 residues) of DnaB also had a critical role in regulating DnaA complex formation at the origin of replication (OriC). This regulatory effect was not obviously observed for DNA substrates containing only two DnaA-boxes. This is the first report showing a physical interaction between DnaA and replicative helicase DnaB from M. tuberculosis and the role in subsequent DnaA-OriC interactions. The findings reported here further the understanding of the regulatory mechanisms for initiation of DNA replication in this important human pathogen.     

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.     

Novel essential residues of Hda for interaction with DnaA in the regulatory inactivation of DnaA: unique roles for Hda AAA+ Box VI and VII motifs

Escherichia coli ATP–DnaA initiates chromosomal replication. For preventing extra‐initiations, a complex of ADP–Hda and the DNA‐loaded replicase clamp promotes DnaA‐ATP hydrolysis, yielding inactive ADP–DnaA. However, the Hda–DnaA interaction mode remains unclear except that the Hda Box VII Arg finger (Arg‐153) and DnaA sensor II Arg‐334 within each AAA⁺ domain are crucial for the DnaA‐ATP hydrolysis. Here, we demonstrate that direct and functional interaction of ADP–Hda with DnaA requires the Hda residues Ser‐152, Phe‐118 and Asn‐122 as well as Hda Arg‐153 and DnaA Arg‐334. Structural analyses suggest intermolecular interactions between Hda Ser‐152 and DnaA Arg‐334 and between Hda Phe‐118 and the DnaA Walker B motif region, in addition to an intramolecular interaction between Hda Asn‐122 and Arg‐153. These interactions likely sustain a specific association of ADP–Hda and DnaA, promoting DnaA‐ATP hydrolysis. Consistently, ATP–DnaA and ADP–DnaA interact with the ADP–Hda‐DNA–clamp complex with similar affinities. Hda Phe‐118 and Asn‐122 are contained in the Box VI region, and their hydrophobic and electrostatic features are basically conserved in the corresponding residues of other AAA⁺ proteins, suggesting a conserved role for Box VI. These findings indicate novel interaction mechanisms for Hda–DnaA as well as a potentially fundamental mechanism in AAA⁺ protein interactions.     

DnaA box sequences as the site for helicase delivery during plasmid RK2 replication initiation in Escherichia coli

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.     

DNA gyrase activity regulates DnaA‐dependent replication initiation in Bacillus subtilis

In bacteria, initiation of DNA replication requires the DnaA protein. Regulation of DnaA association and activity at the origin of replication, oriC, is the predominant mechanism of replication initiation control. One key feature known to be generally important for replication is DNA topology. Although there have been some suggestions that topology may impact replication initiation, whether this mechanism regulates DnaA‐mediated replication initiation is unclear. We found that the essential topoisomerase, DNA gyrase, is required for both proper binding of DnaA to oriC as well as control of initiation frequency in Bacillus subtilis. Furthermore, we found that the regulatory activity of gyrase in initiation is specific to DnaA and oriC. Cells initiating replication from a DnaA‐independent origin, oriN, are largely resistant to gyrase inhibition by novobiocin, even at concentrations that compromise survival by up to four orders of magnitude in oriC cells. Furthermore, inhibition of gyrase does not impact initiation frequency in oriN cells. Additionally, deletion or overexpression of the DnaA regulator, YabA, significantly modulates sensitivity to gyrase inhibition, but only in oriC and not oriN cells. We propose that gyrase is a negative regulator of DnaA‐dependent replication initiation from oriC, and that this regulatory mechanism is required for cell survival.     

DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome.

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single-stranded DNA in a PriA-independent, DnaD- and DnaI-dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.     

NMR structure of the N-terminal domain of the replication initiator protein DnaA

DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 A based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.     

Interactions of DnaA proteins from distantly related bacteria with the replication origin of the broad host range plasmid RK2

Replication initiation of the broad host range plasmid RK2 requires binding of the host-encoded DnaA protein to specific sequences (DnaA boxes) at its replication origin (oriV). In contrast to a chromosomal replication origin, which functionally interacts only with the native DnaA protein of the organism, the ability of RK2 to replicate in a wide range of Gram-negative bacterial hosts requires the interaction of oriV with many different DnaA proteins. In this study we compared the interactions of oriV with five different DnaA proteins. DNase I footprint, gel mobility shift, and surface plasmon resonance analyses showed that the DnaA proteins from Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa bind to the DnaA boxes at oriV and are capable of inducing open complex formation, the first step in the replication initiation process. However, DnaA proteins from two Gram-positive bacteria, Bacillus subtilis and Streptomyces lividans, while capable of specifically interacting with the DnaA box sequences at oriV, do not bind stably and fail to induce open complex formation. These results suggest that the inability of the DnaA protein of a host bacterium to form a stable and functional complex with the DnaA boxes at oriV is a limiting step for plasmid host range.