Primosomal Proteins Research Articles

Screening of genes involved in phage-resistance of Escherichia coli and effects of substances interacting with primosomal protein A on the resistant bacteria.

The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells.

Inhibition of Replication Fork Formation and Progression: Targeting the Replication Initiation and Primosomal Proteins.

Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided.

Characterization of Streptococcus pneumoniae PriA helicase and its ATPase and unwinding activities in DNA replication restart

DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein—a 30 to 50 superfamily-2 DNA helicase—is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities ofStreptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses.

Characterization of the Chimeric PriB-SSBc Protein.

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

SSB Facilitates Fork-Substrate Discrimination by the PriA DNA Helicase.

Primosomal protein A (PriA) is a member of helicase SuperFamily 2. Its role in vivo is to reload the primosome onto resurrected replication forks resulting in the restart of the previously stalled DNA replication process. Single-stranded DNA-binding protein (SSB) plays a key role in mediating activities at replication forks and interacts both physically and functionally with PriA. To gain a mechanistic insight into the PriA–SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriA in vitro in the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates as much as 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for the Escherichia coli protein. SSB, while enhancing the activity of PriA on its preferred fork decreases both the affinity of the helicase for other forks and the catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3′-OH placed at the end of the nascent leading strand at the fork. When both the 3′-OH and SSB are present, the maximum effect on the ATPase activity of the helicase is observed. This ensures that PriA will load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.

Single-molecule binding characterization of primosomal protein PriA involved in replication restart.

DNA damage leads to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal protein PriA specifically recognizes the DNA fork structure and recruits other primosomal proteins to load the replicative helicase, in order to re-establish the replication fork. PriA binding on DNA is the first step to restart replication forks for proper DNA repair. Using a single-molecule fluorescence colocalization experiment, we measured the thermodynamic and real-time kinetic properties of fluorescence-labeled Gram-positive bacteria Geobacillus stearothermophilus PriA binding on DNA forks. We showed that PriA preferentially binds to a DNA fork structure with a fully duplexed leading strand at sub-nanomolar affinity (Kd = 268 ± 99 pM). PriA binds dynamically, and its association and dissociation rate constants can be determined using the appearance and disappearance of the fluorescence signal. In addition, we showed that PriA binds to DNA forks as a monomer using photobleaching step counting. This information offers a molecular basis essential for understanding the mechanism of replication restart.

Characterization of Streptococcus pneumoniae PriA helicase and its ATPase and unwinding activities in DNA replication restart.

DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein - a 3' to 5' superfamily-2 DNA helicase - is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriAdeloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriAdeloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that the presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.

Regulation of DNA Binding and High-Order Oligomerization of the DnaB Helicase Loader.

DnaB is an essential primosomal protein that coloads the replicative helicase in many Gram-positive bacteria, including several human pathogens. Although DnaB is tetrameric in solution, it is from a protein family whose members can oligomerize into large complexes when exposed to DNA. It is currently unknown how DNA binding by DnaB is regulated or how these interactions induce changes in its oligomeric state. Here, we investigated DNA binding by DnaB from Bacillus subtilis and the critical human pathogen Staphylococcus aureus We found that B. subtilis DnaB binds double-stranded DNA as a tetramer; however, M13mp18 single-stranded DNA induces high-order oligomerization. Mutating a conserved motif at the C-terminal end of DnaB stimulates single-stranded DNA binding, suggesting that conformational changes in this region regulate DNA substrate preferences. S. aureus DnaB could also be induced to form high-order oligomers with either M13mp18 or PhiX174 single-stranded DNA. Therefore, oligomeric shifts in DnaB are tightly controlled and this activity is conserved between B. subtilis and a pathogenic species.IMPORTANCE DnaB is a replicative helicase loader involved in initiating DNA replication in many bacterial species. We investigated the binding preferences of DnaB for its DNA substrate and determined that the C-terminal end of the protein plays a critical role in controlling DNA interactions. Furthermore, we found that DNA binding in general did not trigger changes to the oligomeric state of DnaB, but rather, certain types of single-stranded DNA substrates specifically induced DnaB to self-assemble into a large complex. This indicates that the structure of DNA itself is an important regulatory element that influences the behavior of DnaB. Importantly, these observations held for both Bacillus subtilis and the pathogenic species Staphylococcus aureus, demonstrating conserved biochemical functions of DnaB in these species.

Biochemical analysis of PriA Helicases from Gram‐positive Bacteria involved in DNA Replication Restart

DNA replication forks often encounter template DNA lesions, which can stall progression of the replication forks. In gram‐positive bacteria, the PriA‐dependent pathway is the major replication restart mechanism and it requires several primosome proteins. Among them, the PriA protein—a 3′ to 5′ superfamily‐2 DNA helicase—is the key factor recognizing DNA lesions and it further recruits other proteins. Here, we investigated the PriA ATPase and helicase activities of Streptococcus pneumoniae (SpPriA) and Geobacillus stearothermophilus (GstPriA) through biochemical and kinetic analyses. Using various DNA duplexes with unstructured single‐stranded regions, we observed distinct unwinding abilities for GstPriA and SpPriA according to increased GC content of duplex DNA substrate. In contrast to the unwinding activity of GstPriA helicase, we found that SpPriA is unable to unwind duplex DNA substrate with high GC content. However, the presence of DnaD loader protein allows SpPriA to unwind such duplex DNA. Our findings suggest that SpPriA possesses limited unwinding ability in high GC DNA and that DnaD acts as an accessory protein to destabilize stalled replication forks through its single‐stranded DNA binding ability, thereby enhancing PriA unwinding activity.

Single molecule tracking reveals functions for RarA at replication forks but also independently from replication during DNA repair in Bacillus subtilis

RarA is a widely conserved protein proposed to be involved in recombination-dependent replication. We present a cell biological approach to identify functional connections between RarA and other proteins using single molecule tracking. We found that 50% of RarA molecules were static, mostly close to replication forks and likely DNA-bound, while the remaining fraction was highly dynamic throughout the cells. RarA alternated between static and dynamic states. Exposure to H2O2 increased the fraction of dynamic molecules, but not treatment with mitomycin C or with methyl methanesulfonate, which was exacerbated by the absence of RecJ, RecD2, RecS and RecU proteins. The ratio between static and dynamic RarA also changed in replication temperature-sensitive mutants, but in opposite manners, dependent upon inhibition of DnaB or of DnaC (pre)primosomal proteins, revealing an intricate function related to DNA replication restart. RarA likely acts in the context of collapsed replication forks, as well as in conjunction with a network of proteins that affect the activity of the RecA recombinase. Our novel approach reveals intricate interactions of RarA, and is widely applicable for in vivo protein studies, to underpin genetic or biochemical connections, and is especially helpful for investigating proteins whose absence does not lead to any detectable phenotype.

Crystal structure of the C-terminal domain of the primosomal DnaT protein: Insights into a new oligomerization mechanism

DnaT is a replication restart primosomal protein required for re-initiating chromosomal DNA replication in bacteria. DnaT can be a monomer, dimer, trimer, tetramer, or pentamer. The oligomerization and disassembly of DnaT oligomers are critical in primosome assembly. Prior to this work, only the ssDNA-bound structure of the pentameric DnaT truncated protein (aa 84–153; DnaT84–153) was available. The mechanism by which DnaT oligomerizes as different states is unclear. In this paper, we report the crystal structure of the C-terminal domain of DnaT (aa 84–179; DnaTc) at 2.30 Å resolution (PDB entry 6AEQ). DnaTc forms a dimer both in the crystalline state and in solution. As compared with the ssDNA-bound structure of the pentameric DnaT84–153, their subunit–subunit interfaces significantly differ. The different oligomeric architecture suggests a strong conformational change possibly induced by ssDNA. Superposition analysis further indicated that the monomer of a DnaTc dimer shifted away by a distance of 7.5 Å and rotated by an angle of 170° for binding to ssDNA. Basing from these molecular evidence, we discussed and proposed a working model to explain how DnaTc oligomerizes through residue R146 mediation.

Function of a strand-separation pin element in the PriA DNA replication restart helicase

DNA helicases are motor proteins that couple the chemical energy of nucleoside triphosphate hydrolysis to the mechanical functions required for DNA unwinding. Studies of several helicases have identified strand-separating "pin" structures that are positioned to intercept incoming dsDNA and promote strand separation during helicase translocation. However, pin structures vary among helicases and it remains unclear whether they confer a conserved unwinding mechanism. Here, we tested the biochemical and cellular roles of a putative pin element within the Escherichia coli PriA DNA helicase. PriA orchestrates replication restart in bacteria by unwinding the lagging-strand arm of abandoned DNA replication forks and reloading the replicative helicase with the help of protein partners that combine with PriA to form what is referred to as a primosome complex. Using in vitro protein-DNA cross-linking, we localized the putative pin (a β-hairpin within a zinc-binding domain in PriA) near the ssDNA-dsDNA junction of the lagging strand in a PriA-DNA replication fork complex. Removal of residues at the tip of the β-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA β-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted β-hairpin PriA variant displayed wildtype levels of DNA unwinding and PriB binding in vitro These results suggest essential but sequence nonspecific roles for the PriA pin element and coupling of PriA DNA unwinding to its interaction with PriB.

Structural analyses of the bacterial primosomal protein DnaB reveal that it is a tetramer and forms a complex with a primosomal re-initiation protein

The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1-300), from Geobacillus stearothermophilus (GstDnaB1-300) at 2.8 Å resolution. Our structure revealed that GstDnaB1-300 forms a tetramer with two basket-like architectures, a finding consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pulldown assays and analytical ultracentrifugation show that GstDnaB1-300 is sufficient to form a complex with PriA, the primosomal reinitiation protein. Moreover, with the aid of small angle X-ray scattering experiments, we also determined the structural envelope of full-length DnaB (GstDnaBFL) in solution. These small angle X-ray scattering studies indicated that GstDnaBFL has an elongated conformation and that the protruding density envelopes originating from GstDnaB1-300 could completely accommodate the GstDnaB C-terminal domain (residues 301-461). Taken together with biochemical assays, our results suggest that GstDnaB uses different domains to distinguish the PriA interaction and single-stranded DNA binding. These findings can further extend our understanding of primosomal assembly in replication restart.

DnaT is a PriC-binding protein

DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT–PriC changed from red to purple, which indicated the protein–protein interactions due to gold nanoparticle aggregate. In addition, this DnaT–PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10−8 M. These results constitute a pioneering study of the DnaT–PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC–SSB–DNA tricomplex and regulates the PriC-dependent replication restart.

Characterization of Staphylococcus aureus Primosomal DnaD Protein: Highly Conserved C-Terminal Region Is Crucial for ssDNA and PriA Helicase Binding but Not for DnaA Protein-Binding and Self-Tetramerization.

The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD) and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204) showed a stable tetramer in solution. This finding indicates that the C-terminal region aa 196–228 is not crucial for SaDnaD oligomerization. SaDnaD forms distinct complexes with ssDNA of different lengths. In fluorescence titrations, SaDnaD bound to ssDNA with a binding-site size of approximately 32 nt. A stable complex of SaDnaD1-195, SaDnaD1-200, and SaDnaD1-204 with ssDNA dT40 was undetectable, indicating that the C-terminal region of SaDnaD (particularly aa 205–228) is crucial for ssDNA binding. The SPR results revealed that SaDnaD1-195 can interact with SaDnaA but not with SaPriA, which may indicate that DnaD has different binding sites for PriA and DnaA. Both SaDnaD and SaDnaDY176A mutant proteins, but not SaDnaD1-195, can significantly stimulate the ATPase activity of SaPriA. Hence, the stimulation effect mainly resulted from direct contact within the protein—protein interaction, not via the DNA—protein interaction. Kinetic studies revealed that the SaDnaD-SaPriA interaction increases the Vmax of the SaPriA ATPase fivefold without significantly affecting the Km. These results indicate that the conserved C-terminal region is crucial for ssDNA and PriA helicase binding, but not for DnaA protein-binding and self-tetramerization.

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.

Quantitative proteomic analysis of sub-MIC erythromycin inhibiting biofilm formation of S. suis in vitro.

In this study, we identified many proteins involved in cell transport, biological regulation and signal transduction, stress responses and other metabolic processes that were not previously known to be associated with biofilm formation of S. suis and target spot of erythromycin. Therefore, our manuscript represents the most comprehensive analysis of protein profiles of biofilm formation of S. suis inhibited by sub-MIC erythromycin and provides new proteomic information about biofilm formation.

Structure and mechanism of the primosome protein DnaT-functional structures for homotrimerization, dissociation of ssDNA from the PriB·ssDNA complex, and formation of the DnaT·ssDNA complex.

In Escherichia coli, the primosome plays an essential role in replication restart after dissociation of replisomes at the damaged replication fork. As well as PriA and PriB, DnaT, an ssDNA-binding protein, is a key member of the primosome. In this study, limited proteolysis indicated that E. coli DnaT was composed of two domains, consistent with the results of recent studies using Klebsiella pneumonia DnaT. We also found that a specific 24-residue region (Phe42-Asp66) in the N-terminal domain (1-88) was crucial for DnaT trimerization. Moreover, we determined the structure of the DnaT C-terminal domain (89-179) by NMR spectroscopy. This domain included three α-helices and a long flexible C-terminal tail, similar to the C-terminal subdomain of the AAA+ ATPase family. The neighboring histidines, His136 and His137, at a position corresponding to the AAA+ sensor II motif, were suggested to form an ssDNA-binding site. Furthermore, we found that the acidic linker between the two domains had an activity for dissociating ssDNA from the PriB·ssDNA complexes in a manner supported by the conserved acidic residues Asp70 and Glu76. Thus, these findings provide a novel structural basis for understanding the mechanism of DnaT in exposure of ssDNA and reloading of the replicative helicase at the stalled replication fork. The coordinates used for the ensemble of NMR structures have been deposited in the Protein Data Bank under accession code 2ru8. The NMR data have been deposited in the BioMagResBank (www.bmrb.wisc.edu) under accession number 11549.

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 DnaT84–153-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.

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein.

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.