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.     

Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.     

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.     

Cellular characterization of the primosome and rep helicase in processing and restoration of replication following arrest by UV-induced DNA damage in Escherichia coli

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion.     

Primosome assembly site in Bacillus subtilis.

A single-strand initiation site was detected on the Enterococcus faecalis plasmid pAM beta 1 by its ability to prevent accumulation of single stranded DNA of a rolling circle plasmid, both in Bacillus subtilis and Staphylococcus aureus. This site, designated ssiA, is located on the lagging strand template, approximately 150 bp downstream from the replication origin. ssiA priming activity requires the DnaE primase, the DnaC replication fork helicase, as well as the products of the dnaB, dnaD and dnaI genes of B.subtilis, but not the RNA polymerase. The primase and the replication fork helicase requirements indicate that ssiA is a primosome assembly site. Interestingly, the pAM beta 1 lagging strand synthesis is inefficient when any of the proteins involved in ssiA activity is mutated, but occurs efficiently in the absence of ssiA. This suggests that normal plasmid replication requires primosome assembly and that the primosome can assemble not only at ssiA but also elsewhere on the plasmid. This work for the first time describes a primosome in a Gram-positive bacterium. Involvement of the B.subtilis proteins DnaB, DnaD and DnaI, which do not have any known analogue in Escherichia coli, raises the possibility that primosome assembly and/or function in B.subtilis differs from that in E.coli.     

Escherichia coli PriA protein, two modes of DNA binding and activation of ATP hydrolysis

Escherichia coli PriA protein plays crucial roles in processing of arrested replication forks. PriA serves as a sensor/stabilizer for an arrested replication fork and eventually promotes restart of DNA replication through assembly of a primosome. PriA carries a 3' terminus binding pocket required for its high affinity binding to a specific arrested fork as well as for its biological functions. We show here that PriA binds to DNA in a manner either dependent on or independent of 3' terminus recognition. The former mode of binding requires the 3' terminus binding pocket present at the N-terminal half of the 181-residue DNA binding domain and exhibits specific bipartite interaction on the template DNA. The latter mode is independent of the pocket function, but requires the C-terminal half of the same domain. ATP hydrolysis activity of PriA can be stimulated in vitro by either of the two binding modes. We propose architecture of PriA bound to various arrested replication fork structures and discuss its implication in helicase activation and ATP hydrolysis.     

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.     

Dissecting the functional role of PriA protein-catalysed primosome assembly in Escherichia coli DNA replication

The multi-functional PriA protein of Escherichia coli (formerly replication factor Y or protein n') serves to guide the ordered assembly of the primosome, a mobile multiprotein replication priming/helicase complex. Primosome assembly is essential for bacteriophage OX174 complementary DNA strand synthesis and ColE1-type plasmid replication reconstituted in vitro with purified proteins. The biochemical activities of the primosome suggest that it can fulfill the primase/helicase requirement on the lagging-strand DNA template during cellular DNA replication. However, reconstruction in vitro of DNA replication of small plasmids containing the E. coli origin of DNA replication (oriC) does not require the complete complement of primosomal proteins. Thus, the extent to which PriA-catalysed primosome assembly participates in chromosomal replication has remained unclear. The recent isolation of the genes encoding PriA, PriB (protein n), PriC (protein n"), and DnaT (protein i) has provided the necessary tools for addressing this issue. The phenotype of mutations in these genes, and other results described in this review, suggest that assembly of the primosome catalysed by PriA does in fact contribute at some stage to normal cellular DNA replication. A model for primososme-catalysed reactivation of a dysfunctional replication fork is discussed.     

Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

When replication stalls and forks disassemble, the restart primosome is required to reload the replicative helicase so that chromosomal replication can be reinitiated. We have taken a photo-cross-linking approach, using model replication forks containing a phenyl diazirine placed at single locations, to determine the positions of primosomal protein binding and changes in interactions that occur during the assembly reaction. This approach revealed a novel mode for single-stranded DNA-binding protein (SSB)-DNA binding, in which SSB interacts with both the leading and lagging single-strand segments and the parental duplex of the fork. Cross-linking to a novel region within SSB is observed only when it is bound to forked structures. This binding mode is also followed by PriB. PriA binds to the fork, excluding SSB and PriB, interacting with the primer terminus, single-stranded leading and lagging strands and duplex in immediate proximity of the fork. SSB binds to flanking single-stranded segments distal to the fork in the presence of PriA. The addition of PriB or DnaT to a PriA-SSB-fork complex does not lead to cross-linking or displacement, suggesting that their association is through protein-protein interactions at early stages of the reaction. Upon addition of DnaC and the DnaB helicase in the presence of ATPγS, helicase is assembled, leading to contacts within the duplex region on the tracking (lagging) strand and strong contacts with the displaced leading single strand near the fork. PriA is displaced from DNA upon helicase assembly.     

PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells

The Escherichia coli PriA protein loads the DnaB replicative helicase at branched DNA structures independently of the replication initiator protein, DnaA, and thereby facilitates assembly of functional replisomes at sites removed from oriC. It is therefore a critical factor in the rescue of replication forks stalled at DNA lesions. It is also a DNA helicase. We describe insertions near the 3' end of priA that interfere with PriA activity. These insertions and the previously described priA300 encoding helicase-defective PriA K230R are shown to be effective suppressors of the DNA repair defect in recG strains, but substantially reduce the ability of ruv mutants to survive DNA damage. The data presented suggest that PriA helicase in conjunction with RecG can promote direct rescue of stalled forks independently of the recombinational pathway promoted by the combined activities of the RuvABC, RecBCD and RecA proteins, which requires only the primosome assembly activity of PriA to load DnaB at D loops. In cells lacking the helicase activity of PriA, we propose that stalled forks can be redirected to the recombination pathway via a Holliday junction intermediate common to both pathways, thus explaining the resistance of these cells to DNA damage.     

A hand-off mechanism for primosome assembly in replication restart

Collapsed DNA replication forks must be reactivated through origin-independent reloading of the replication machinery (replisome) to ensure complete duplication of cellular genomes. In E. coli, the PriA-dependent pathway is the major replication restart mechanism and requires primosome proteins PriA, PriB, and DnaT for replisome reloading. However, the molecular mechanisms that regulate origin-independent replisome loading are not fully understood. Here, we demonstrate that assembly of primosome protein complexes represents a key regulatory mechanism, as inherently weak PriA-PriB and PriB-DnaT interactions are strongly stimulated by single-stranded DNA. Furthermore, the binding site on PriB for single-stranded DNA partially overlaps the binding sites for PriA and DnaT, suggesting a dynamic primosome assembly process in which single-stranded DNA is handed off from one primosome protein to another as a repaired replication fork is reactivated. This model helps explain how origin-independent initiation of DNA replication is restricted to repaired replication forks, preventing overreplication of the genome.     

PriB stimulates PriA helicase via an interaction with single-stranded DNA

The frequency with which replication forks break down in all organisms requires that specific mechanisms ensure completion of genome duplication. In Escherichia coli a major pathway for reloading of the replicative apparatus at sites of fork breakdown is dependent on PriA helicase. PriA acts in conjunction with PriB and DnaT to effect loading of the replicative helicase DnaB back onto the lagging strand template, either at stalled fork structures or at recombination intermediates. Here we showed that PriB stimulates PriA helicase, acting to increase the apparent processivity of PriA. This stimulation correlates with the ability of PriB to form a ternary complex with PriA and DNA structures containing single-stranded DNA, suggesting that the known single-stranded DNA binding function of PriB facilitates unwinding by PriA helicase. This enhanced apparent processivity of PriA might play an important role in generating single-stranded DNA at stalled replication forks upon which to load DnaB. However, stimulation of PriA by PriB is not DNA structure-specific, demonstrating that targeting of stalled forks and recombination intermediates during replication restart likely resides with PriA alone.     

Stabilization of a stalled replication fork by concerted actions of two helicases

PriA helicase plays crucial roles in restoration of arrested replication forks. It carries a "3' terminus binding pocket" in its N-terminal DNA binding domain, which is required for high affinity binding of PriA to a fork carrying a 3'-end of a nascent leading strand at the branch. We show that the abrogation of the 3' terminus recognition either by a mutation in the 3' terminus binding pocket or by the bulky modification of the 3'-end leads to unwinding of the unreplicated duplex arm on this fork, causing potential fork destabilization. This indicates a critical role of the 3' terminus binding pocket of PriA in its "stable" binding at the fork for primosome assembly. In contrast, PriA unwinds the unreplicated duplex region on a fork without a 3'-end, potentially destabilizing the fork. However, this process is inhibited by RecG helicase, capable of regressing the fork until the 3'-end of the nascent leading strand reaches the branch. PriA now stably binds to this regressed fork, stabilizing it. Using a model arrest-fork-substrate, we reconstitute the above process in vitro with RecG and PriA proteins. Our results present a novel mechanism by which two helicases function in a highly coordinated manner to generate a structure in which an arrested fork is stabilized for further repair and/or replication restart.     

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.     

PriA-directed assembly of a primosome on D loop DNA.

Escherichia coli strains carrying null mutations in priA are chronically induced for the SOS response and are defective in homologous recombination, repair of UV damaged DNA, double-strand break repair, and both induced and constitutive stable DNA replication. This led to the proposal that PriA directed replication fork assembly at D loops formed by the homologous recombination machinery. The demonstration that PriA specifically recognized and bound D loop DNA supported this hypothesis. Using DNA footprinting as an assay, we show here that PriA also directs the assembly of a varphiX174-type primosome on D loop DNA. The ability to load a complete primosome on D loop DNA is a step necessary for replication fork assembly.     

Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities

The PriA protein of Escherichia coli plays a key role in the rescue of replication forks stalled on the template DNA. One attractive model of rescue relies on homologous recombination to establish a new fork via PriA-mediated loading of the DnaB replicative helicase at D loop intermediates. We provide genetic and biochemical evidence that PriA helicase activity can also rescue a stalled fork by an alternative mechanism that requires manipulation of the fork before loading of DnaB on the lagging strand template. This direct rescue depends on RecG, which unwinds forks and Holliday junctions and interconverts these structures. The combined action of PriA and RecG helicase activities may thus avoid the potential dangers of rescue pathways involving fork breakage and recombination.     

Crystal structure of PriB, a component of the Escherichia coli replication restart primosome

Maintenance of genome stability following DNA damage requires origin-independent reinitiation of DNA replication at repaired replication forks. In E. coli, PriA, PriB, PriC, and DnaT play critical roles in recognizing repaired replication forks and reloading the replisome onto the template to reinitiate DNA replication. Here, we report the 2.0 A resolution crystal structure of E. coli PriB, revealing a dimer that consists of a single structural domain formed by two oligonucleotide/oligosaccharide binding (OB) folds. Structural similarity of PriB to single-stranded DNA binding proteins reveals insights into its mechanisms of DNA binding. The structure further establishes a putative protein interaction surface that may contribute to the role of PriB in primosome assembly by facilitating interactions with PriA and DnaT. This is the first high-resolution structure of a protein involved in oriC-independent replisome loading and provides unique insight into mechanisms of replication restart in E. coli.     

The disposition of nascent strands at stalled replication forks dictates the pathway of replisome loading during restart

Rescue of arrested and collapsed replication forks is essential for maintenance of genomic integrity. One system for origin of replication-independent loading of the DnaB replicative helicase and subsequent replisome reassembly requires the structure-specific recognition factor PriA and the assembly factors PriB and DnaT. Here, we provide biochemical evidence for an alternate system for DnaB loading that requires only PriC. Furthermore, the choice of which system is utilized during restart is dictated by the nature of the structure of the stalled replication fork. PriA-dependent reactions are most robust on fork structures with no gaps in the leading strand, such as is found at the junction of a D loop, while the PriC-dependent system preferentially utilizes fork structures with large gaps in the leading strand. These observations suggest that the type of initial damage on the DNA template and how the inactivated fork is processed ultimately influence the choice of enzymatic restart pathway.     

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition.

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.     

Crystal structure of DnaT84–153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode

DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT^84–153-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.