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.     

Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex

Structural details of initiator proteins for DNA replication have provided clues to the molecular events in this process. EM reconstructions of the Drosophila melanogaster origin recognition complex (ORC) reveal nucleotide-dependent conformational changes in the core of the complex. All five AAA+ domains in ORC contain a conserved structural element that, in DnaA, promotes formation of a right-handed helix, indicating that helical AAA+ substructures may be a feature of all initiators. A DnaA helical pentamer can be docked into ORC, and the location of Orc5 uniquely positions this core. The results suggest that ATP-dependent conformational changes observed in ORC derive from reorientation of the AAA+ domains. By analogy to the DNA-wrapping activity of DnaA, we posit that ORC together with Cdc6 prepares origin DNA for helicase loading through mechanisms related to the established pathway of prokaryotes.     

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.     

Topological characterization of the DnaA-oriC complex using single-molecule nanomanipuation

In most bacteria, the timing and synchrony of initiation of chromosomal replication are determined by the binding of the AAA(+) protein DnaA to a set of high- and low-affinity sites found within the origin of chromosomal replication (oriC). Despite the large amount of information on the role and regulation of DnaA, the actual structure of the DnaA-oriC complex and the mechanism by which it primes the origin for the initiation of replication remain unclear. In this study, we have performed magnetic tweezers experiments to investigate the structural properties of the DnaA-oriC complex. We show that the DnaA-ATP-oriC complex adopts a right-handed helical conformation involving a variable amount of DNA and protein whose features fit qualitatively as well as quantitatively with an existing model based on the crystal structure of a truncated DnaA tetramer obtained in the absence of DNA. We also investigate the topological effect of oriC's DNA unwinding element.     

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.     

AT-rich region and repeated sequences - the essential elements of replication origins of bacterial replicons

Repeated sequences are commonly present in the sites for DNA replication initiation in bacterial, archaeal, and eukaryotic replicons. Those motifs are usually the binding places for replication initiation proteins or replication regulatory factors. In prokaryotic replication origins, the most abundant repeated sequences are DnaA boxes which are the binding sites for chromosomal replication initiation protein DnaA, iterons which bind plasmid or phage DNA replication initiators, defined motifs for site-specific DNA methylation, and 13-nucleotide-long motifs of a not too well-characterized function, which are present within a specific region of replication origin containing higher than average content of adenine and thymine residues. In this review, we specify methods allowing identification of a replication origin, basing on the localization of an AT-rich region and the arrangement of the origin's structural elements. We describe the regularity of the position and structure of the AT-rich regions in bacterial chromosomes and plasmids. The importance of 13-nucleotide-long repeats present at the AT-rich region, as well as other motifs overlapping them, was pointed out to be essential for DNA replication initiation including origin opening, helicase loading and replication complex assembly. We also summarize the role of AT-rich region repeated sequences for DNA replication regulation.     

Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre-replicative complex (pre-RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high- and low-affinity recognition sites within the unique oriC locus, comprises the pre-RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher-order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low-affinity DnaA binding sites. As DnaA levels increase, peripheral high-affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre-RC activity and may be a feature found in all bacterial replication origins.     

The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation

The initiation of DNA replication is a key event in the cell cycle of all organisms. In bacteria, replication initiation occurs at specific origin sequences that are recognized and processed by an oligomeric complex of the initiator protein DnaA. We have determined the structure of the conserved core of the Aquifex aeolicus DnaA protein to 2.7 A resolution. The protein comprises an AAA+ nucleotide-binding fold linked through a long, helical connector to an all-helical DNA-binding domain. The structure serves as a template for understanding the physical consequences of a variety of DnaA mutations, and conserved motifs in the protein suggest how two critical aspects of origin processing, DNA binding and homo-oligomerization, are mediated. The spatial arrangement of these motifs in DnaA is similar to that of the eukaryotic-like archaeal replication initiation factor Cdc6/Orc1, demonstrating that mechanistic elements of origin processing may be conserved across bacterial, archaeal and eukaryotic domains of life.     

Formation of an ATP-DnaA-specific initiation complex requires DnaA Arginine 285, a conserved motif in the AAA+ protein family

Escherichia coli DnaA protein, a member of the AAA+ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaA-specific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATP-DnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the Arg-285 residue for initiation. Sequence and structural analyses suggest that the DnaA Arg-285 residue is an arginine finger, an AAA+ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA Arg-285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaA-specific binding sites in the complex.     

A Structural Basis for the Regulatory Inactivation of DnaA

Regulatory inactivation of DnaA is dependent on Hda (homologous to DnaA), a protein homologous to the AAA+ (ATPases associated with diverse cellular activities) ATPase region of the replication initiator DnaA. When bound to the sliding clamp loaded onto duplex DNA, Hda can stimulate the transformation of active DnaA-ATP into inactive DnaA-ADP. The crystal structure of Hda from Shewanella amazonensis SB2B at 1.75 Å resolution reveals that Hda resembles typical AAA+ ATPases. The arrangement of the two subdomains in Hda (residues 1-174 and 175-241) differs dramatically from that of DnaA. A CDP molecule anchors the Hda domains in a conformation that promotes dimer formation. The Hda dimer adopts a novel oligomeric assembly for AAA+ proteins in which the arginine finger, crucial for ATP hydrolysis, is fully exposed and available to hydrolyze DnaA-ATP through a typical AAA+ type of mechanism. The sliding clamp binding motifs at the N-terminus of each Hda monomer are partially buried and combine to form an antiparallel β-sheet at the dimer interface. The inaccessibility of the clamp binding motifs in the CDP-bound structure of Hda suggests that conformational changes are required for Hda to form a functional complex with the clamp. Thus, the CDP-bound Hda dimer likely represents an inactive form of Hda.     

AAA+ ATPases in the Initiation of DNA Replication

All cellular organisms and many viruses rely on large, multi-subunit molecular machines, termed replisomes, to ensure that genetic material is accurately duplicated for transmission from one generation to the next. Replisome assembly is facilitated by dedicated initiator proteins, which serve to both recognize replication origins and recruit requisite replisomal components to the DNA in a cell-cycle coordinated manner. Exactly how imitators accomplish this task, and the extent to which initiator mechanisms are conserved among different organisms have remained outstanding issues. Recent structural and biochemical findings have revealed that all cellular initiators, as well as the initiators of certain classes of double-stranded DNA viruses, possess a common adenine nucleotide-binding fold belonging to the ATPases Associated with various cellular Activities (AAA+) family. This review focuses on how the AAA+ domain has been recruited and adapted to control the initiation of DNA replication, and how the use of this ATPase module underlies a common set of initiator assembly states and functions. How biochemical and structural properties correlate with initiator activity, and how species-specific modifications give rise to unique initiator functions, are also discussed.     

AAA+ ATPases in the initiation of DNA replication

All cellular organisms and many viruses rely on large, multi-subunit molecular machines, termed replisomes, to ensure that genetic material is accurately duplicated for transmission from one generation to the next. Replisome assembly is facilitated by dedicated initiator proteins, which serve to both recognize replication origins and recruit requisite replisomal components to the DNA in a cell-cycle coordinated manner. Exactly how imitators accomplish this task, and the extent to which initiator mechanisms are conserved among different organisms have remained outstanding issues. Recent structural and biochemical findings have revealed that all cellular initiators, as well as the initiators of certain classes of double-stranded DNA viruses, possess a common adenine nucleotide-binding fold belonging to the ATPases Associated with various cellular Activities (AAA+) family. This review focuses on how the AAA+ domain has been recruited and adapted to control the initiation of DNA replication, and how the use of this ATPase module underlies a common set of initiator assembly states and functions. How biochemical and structural properties correlate with initiator activity, and how species-specific modifications give rise to unique initiator functions, are also discussed.     

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.     

Bacterial initiators form dynamic filaments on single-stranded DNA monomer by monomer

DNA replication initiation is mediated across all domains of life by initiator proteins oligomerizing at replication origins. Recently, it was shown that initiators can directly bind single-stranded DNA (ssDNA) and thus might enhance origin melting. In this study, we used single-molecule fluorescence assays to probe the ssDNA binding mechanism of the replication initiator DnaA. Our experiments revealed that DnaA forms a dynamic filament on ssDNA in 3′ to 5′ directionality in the presence of ATP and analogs. After nucleation with a three-monomer seed, monomers dynamically assemble and disassemble one monomer at a time at the 5′ end, each monomer binding three nucleotides of ssDNA. The addition of adjacent double-stranded DnaA binding sites stabilized the DnaA filament on ssDNA. Our results extend the current models of origin melting via DnaA ssDNA interaction.     

Effects of purified SeqA protein on oriC-dependent DNA replication in vitro.

In vivo studies suggest that the Escherichia coli SeqA protein modulates replication initiation in two ways: by delaying initiation and by sequestering newly replicated origins from undergoing re-replication. As a first approach towards understanding the biochemical bases for these effects, we have examined the effects of purified SeqA protein on replication reactions performed in vitro on an oriC plasmid. Our results demonstrate that SeqA directly affects the biochemical events occurring at oriC. First, SeqA inhibits formation of the pre-priming complex. Secondly, SeqA can inhibit replication from an established pre-priming complex, without disrupting the complex. Thirdly, SeqA alters the dependence of the replication system on DnaA protein concentration, stimulating replication at low concentrations of DnaA. Our data suggest that SeqA participates in the assembly of initiation-competent complexes at oriC and, at a later stage, influences the behaviour of these complexes.     

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.     

A critical DnaA box directs the cooperative binding of the Escherichia coli DnaA protein to the plasmid RK2 replication origin.

The requirement of DnaA protein binding for plasmid RK2 replication initiation the Escherichia coli was investigated by constructing mutations in the plasmid replication origin that scrambled or deleted each of the four upstream DnaA boxes. Altered origins were analyzed for replication activity in vivo and in vitro and for binding to the E. coli DnaA protein using a gel mobility shift assay and DNase I footprinting. Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein binding to all four boxes. Unlike DnaA binding to the E. coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative with box 4 acting as the "organizer" for the formation of the DnaA-oriV nucleoprotein complex. Interestingly, the inversion of box 4 also abolished replication activity, but did not result in a loss of binding to the other boxes. However, DnaA binding to this mutant origin was no longer cooperative. These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication.     

A comprehensive set of DnaA-box mutations in the replication origin, oriC, of Escherichia coli

We probed the complex between the replication origin, oriC , and the initiator protein DnaA using different types of mutations in the five binding sites for DnaA, DnaA boxes R1–R4 and M: (i) point mutations in individual DnaA boxes and combinations of them; (ii) replacement of the DnaA boxes by a scrambled 9 bp non-box motif; (iii) positional exchange; and (iv) inversion of the DnaA boxes. For each of the five DnaA boxes we found at least one type of mutation that resulted in a phenotype. This demonstrates that all DnaA boxes in oriC have a function in the initiation process. Most mutants with point mutations retained some origin activity, and the in vitro DnaA-binding capacity of these origins correlated well with their replication proficiency. Inversion or scrambling of DnaA boxes R1 or M inactivated oriC -dependent replication of joint replicons or minichromosomes under all conditions, demonstrating the importance of these sites. In contrast, mutants with inverted or scrambled DnaA boxes R2 or R4 could not replicate in wild-type hosts but gave transformants in host strains with deleted or compromised chromosomal oriC at elevated DnaA concentrations. We conclude that these origins require more DnaA per origin for initiation than does wild-type oriC . Mutants in DnaA box R3 behaved essentially like wild-type oriC , except for those in which the low-affinity box R3 was replaced by the high-affinity box R1. Apparently, initiation is possible without DnaA binding to box R3, but high-affinity DnaA binding to DnaA box R3 upsets the regulation. Taken together, these results demonstrate that there are finely tuned DnaA binding requirements for each of the individual DnaA boxes for optimal build-up of the initiation complex and replication initiation in vivo     

The box VII motif of Escherichia coli DnaA protein is required for DnaA oligomerization at the E. coli replication origin

Escherichia coli DnaA protein initiates DNA replication from the chromosomal origin, oriC, and regulates the frequency of this process. Structure-function studies indicate that the replication initiator comprises four domains. Based on the structural similarity of Aquifex aeolicus DnaA to other AAA+ proteins that are oligomeric, it was proposed that Domain III functions in oligomerization at oriC (Erzberger, J. P., Pirruccello, M. M., and Berger, J. M. (2002) EMBO J. 21, 4763-4773). Because the Box VII motif within Domain III is conserved among DnaA homologues and may function in oligomerization, we substituted conserved Box VII amino acids of E. coli DnaA with alanine by site-directed mutagenesis to examine the role of this motif. All mutant proteins are inactive in initiation from oriC in vivo and in vitro, but they support RK2 plasmid DNA replication in vivo. Thus, RK2 requires only a subset of DnaA functions for plasmid DNA replication. Biochemical studies on a mutant DnaA carrying an alanine substitution at arginine 281 (R281A) in Box VII show that it is inactive in in vitro replication of an oriC plasmid, but this defect is not from the failure to bind to ATP, DnaB in the DnaB-DnaC complex, or oriC. Because the mutant DnaA is also active in the strand opening of oriC, whereas DnaB fails to bind to this unwound region, the open structure is insufficient by itself to load DnaB helicase. Our results show that the mutant fails to form a stable oligomeric DnaA-oriC complex, which is required for the loading of DnaB.