Primosomal Proteins Research Articles

Structural insight into the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.

Replication restart primosome is a complex dynamic system that is essential for bacterial survival. This system uses various proteins to reinitiate chromosomal DNA replication to maintain genetic integrity after DNA damage. The replication restart primosome in Escherichia coli is composed of PriA helicase, PriB, PriC, DnaT, DnaC, DnaB helicase, and DnaG primase. The assembly of the protein complexes within the forked DNA responsible for reloading the replicative DnaB helicase anywhere on the chromosome for genome duplication requires the coordination of transient biomolecular interactions. Over the last decade, investigations on the structure and mechanism of these nucleoproteins have provided considerable insight into primosome assembly. In this review, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.

The N-terminal domain of DnaT, a primosomal DNA replication protein, is crucial for PriB binding and self-trimerization

DnaT and PriB are replication restart primosomal proteins required for re-initiating chromosomal DNA replication in bacteria. Although the interaction of DnaT with PriB has been proposed, which region of DnaT is involved in PriB binding and self-trimerization remains unknown. In this study, we identified the N-terminal domain in DnaT (aa 1–83) that is important in PriB binding and self-trimerization but not in single-stranded DNA (ssDNA) binding. DnaT and the deletion mutant DnaT42-179 protein can bind to PriB according to native polyacrylamide gel electrophoresis, Western blot analysis, and pull-down assay, whereas DnaT84-179 cannot bind to PriB. In contrast to DnaT, DnaT26-179, and DnaT42-179 proteins, which form distinct complexes with ssDNA of different lengths, DnaT84-179 forms only a single complex with ssDNA. Analysis of DnaT84-179 protein by gel filtration chromatography showed a stable monomer in solution rather than a trimer, such as DnaT, DnaT26-179, and DnaT42-179 proteins. These results constitute a pioneering study of the domain definition of DnaT. Further research can directly focus on determining how DnaT binds to the PriA–PriB–DNA tricomplex in replication restart by the hand-off mechanism.

DnaT is a single‐stranded DNA binding protein

DnaT is one of the replication restart primosomal proteins required for reinitiating chromosomal DNA replication in bacteria. In this study, we identified and characterized the single-stranded DNA (ssDNA)-binding properties of DnaT using electrophoretic mobility shift analysis (EMSA), bioinformatic tools and two deletion mutant proteins, namely, DnaT26-179 and DnaT42-179. ConSurf analysis indicated that the N-terminal region of DnaT is highly variable. The analysis of purified DnaT and the deletion mutant protein DnaT42-179 by gel filtration chromatography showed a stable trimer in solution, indicating that the N-terminal region, amino acid 1-41, is not crucial for the oligomerization of DnaT. Contrary to PriB, which forms a single complex with a series of ssDNA homopolymers, DnaT, DnaT26-179 and DnaT42-179 form distinct complexes with ssDNA of different lengths and the size of binding site of 26±2 nucleotides (nt). Using bioinformatic programs (ps)(2) and the analysis of the positively charged/hydrophobic residue distribution, as well as the biophysical results in this study, we propose a binding model for the DnaT trimer-ssDNA complex, in which 25-nt-long ssDNA is tethered on the surface groove located in the highly conserved C-terminal domain of DnaT. These results constitute the first study regarding ssDNA-binding activity of DnaT. Consequently, a hand-off mechanism for primosome assembly was modified.

Yeast Two-Hybrid Analysis of PriB-Interacting Proteins in Replication Restart Primosome: A Proposed PriB–SSB Interaction Model

PriB is one of the components of the replication restart primosome. The activity mediated by the primosomal proteins, including PriB, is required for reinitiating chromosomal DNA replication in bacteria after DNA damage. As such, the study of the interactions between PriB and members in the primosome is essential to better understand their mechanism of action. In this study, we investigated PriB-interacting proteins in the primosome through the yeast two-hybrid system. Yeast two-hybrid analysis using two strains, Y187 and AH109, revealed that PriB interacts with PriA, DnaT, SSB, and itself and does not interact with DnaA, DnaB, DnaC, or DnaG. In addition, mutational analysis showed that PriB may bind to SSB via K82, K84, and K89 in the L(45) loop. Based on these preliminary data, we proposed a PriB-SSB interaction model. Further work can focus on determination of how PriB binds to the PriA-SSB complex in replication restart.

Indispensible Replication Restart Helicase PriA Aids Bacterial Survival: A SMART Team Story

Approximately one disruption in DNA replication occur every cell cycle in bacteria leading to partially duplicated chromosomes. Since unfinished replication can result in genome instability and cell death, bacteria need a mechanism to reload the replication machinery onto the genome. Known as the replication restart primosome (RRP), several proteins function to reload the replicative helicase onto abandoned replication forks, restarting DNA replication. PriA is the most conserved member of the RRP, initiating the dominant replication restart pathway. A helicase, PriA remodels collapsed forks and serves as a platform for binding of other primosomal proteins. The Madison West High School Students Modeling a Research Topic (SMART) Team modeled PriA using 3D printing technology. PriA is a multi‐domain protein and residues important for DNA binding, ATP hydrolysis, and helicase activity are modeled. Since DNA replication restart pathways are essential in preserving genomic integrity and cell viability in bacteria, studies of PriA offer an approach to developing novel antibacterial compounds. Supported by a grant from NIH‐CTSA.

The Escherichia coli primosomal DnaT protein exists in solution as a monomer-trimer equilibrium system.

The oligomerization reaction of the Escherichia coli DnaT protein has been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. In solution, DnaT exists as a monomer-trimer equilibrium system. At the estimated concentration in the E. coli cell, DnaT forms a mixture of the monomer and trimer states with a 3:1 molar ratio. In spite of the modest affinity, the trimerization is a highly cooperative process, without the detectable presence of the intervening dimer. The DnaT monomer consists of a large N-terminal core domain and a small C-terminal region. The removal of the C-terminal region dramatically affects the oligomerization process. The isolated N-terminal domain forms a dimer instead of the trimer. These results indicate that the DnaT monomer possesses two structurally different, interacting sites. One site is located on the N-terminal domain, and two monomers, in the trimer, are associated through their binding sites located on that domain. The C-terminal region forms the other interacting site. The third monomer is engaged through the C-terminal regions. Surprisingly, the high affinity of the N-terminal domain dimer indicates that the DnaT monomer undergoes a conformational transition upon oligomerization, involving the C-terminal region. These data and the high specificity of the trimerization reaction, i.e., lack of any oligomers higher than a trimer, indicate that each monomer in the trimer is in contact with the two remaining monomers. A model of the global structure of the DnaT trimer based on the thermodynamic and hydrodynamic data is discussed.

Crystal structure and DNA‐binding mode of Klebsiella pneumoniae primosomal PriB protein

PriB is a primosomal DNA replication protein required for the re-initiation of replication in bacteria. In this study, we investigated the gene expression of PriB in Klebsiella pneumoniae (KpPriB) and characterized the gene product through crystal structural and functional analyses. Quantitative polymerase chain reaction analysis (Q-PCR) indicated that the 104-aa priB was expressed in K. pneumoniae with a C(T) value of 22.4. The crystal structure of KpPriB (Protein Data Bank entry: 4APV) determined at a resolution of 2.1 Å was similar to that of Escherichia coli PriB (EcPriB). KpPriB formed a single complex with single-stranded DNA (ssDNA) of different lengths, suggesting a highly cooperative process. Structure-based mutational analysis revealed that substitution at K18, F42, R44, W47, K82, K84, or K89 but not R34 in KpPriB had a significant effect on both ssDNA and double-stranded DNA (dsDNA) binding. Based on these findings, the known ssDNA interaction sites of PriB were expanded to include R44 and F42, thus allowing nucleic acids to wrap around the whole PriB protein.

The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis

DnaA is an AAA+ ATPase and the conserved replication initiator in bacteria. Bacteria control the timing of replication initiation by regulating the activity of DnaA. DnaA binds to multiple sites in the origin of replication (oriC) and is required for recruitment of proteins needed to load the replicative helicase. DnaA also binds to other chromosomal regions and functions as a transcription factor at some of these sites. Bacillus subtilis DnaD is needed during replication initiation for assembly of the replicative helicase at oriC and during replication restart at stalled replication forks. DnaD associates with DnaA at oriC and at other chromosomal regions bound by DnaA. Using purified proteins, we found that DnaD inhibited the ability of DnaA to bind cooperatively to DNA and caused a decrease in the apparent dissociation constant. These effects of DnaD were independent of the ability of DnaA to bind or hydrolyze ATP. Other proteins known to regulate B. subtilis DnaA also affect DNA binding, whereas much of the regulation of Escherichia coli DnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange for B. subtilis DnaA was high and not affected by DnaD. The rapid exchange is similar to that of Staphylococcus aureus DnaA and in contrast to the low exchange rate of Escherichia coli DnaA. We suggest that organisms in which DnaA has a high rate of nucleotide exchange predominantly regulate the DNA binding activity of DnaA and that those with low rates of exchange regulate hydrolysis and exchange.

Chromosomal replication initiation machinery of low-G+C-content Firmicutes.

Much of our knowledge of the initiation of DNA replication comes from studies in the gram-negative model organism Escherichia coli. However, the location and structure of the origin of replication within the E. coli genome and the identification and study of the proteins which constitute the E. coli initiation complex suggest that it might not be as universal as once thought. The archetypal low-G+C-content gram-positive Firmicutes initiate DNA replication via a unique primosomal machinery, quite distinct from that seen in E. coli, and an examination of oriC in the Firmicutes species Bacillus subtilis indicates that it might provide a better model for the ancestral bacterial origin of replication. Therefore, the study of replication initiation in organisms other than E. coli, such as B. subtilis, will greatly advance our knowledge and understanding of these processes as a whole. In this minireview, we highlight the structure-function relationships of the Firmicutes primosomal proteins, discuss the significance of their oriC architecture, and present a model for replication initiation at oriC.

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.

Cell Size Control in Bacteria

Like eukaryotes, bacteria must coordinate division with growth to ensure cells are the appropriate size for a given environmental condition or developmental fate. As single-celled organisms, nutrient availability is one of the strongest influences on bacterial cell size. Classic physiological experiments conducted over four decades ago first demonstrated that cell size is directly correlated with nutrient source and growth rate in the Gram-negative bacterium Salmonella typhimurium. This observation subsequently served as the basis for studies revealing a role for cell size in cell cycle progression in a closely related organism, Escherichia coli. More recently, the development of powerful genetic, molecular, and imaging tools has allowed us to identify and characterize the nutrient-dependent pathway responsible for coordinating cell division and cell size with growth rate in the Gram-positive model organism Bacillus subtilis. Here, we discuss the role of cell size in bacterial growth and development and propose a broadly applicable model for cell size control in this important and highly divergent domain of life.

Replication Fork Reactivation in a dnaC2 Mutant at Non-Permissive Temperature in Escherichia coli

Replicative helicases unwind double-stranded DNA in front of the polymerase and ensure the processivity of DNA synthesis. In Escherichia coli, the helicase loader DnaC as well as factors involved in the formation of the open complex during the initiation of replication and primosomal proteins during the reactivation of arrested replication forks are required to recruit and deposit the replicative helicase onto single-stranded DNA prior to the formation of the replisome. dnaC2 is a thermosensitive allele of the gene specifying the helicase loader; at non-permissive temperature replication cannot initiate, but most ongoing rounds of replication continues through to completion (18% of dnaC2 cells fail to complete replication at non-permissive temperature). An assumption, which may be drawn from this observation, is that only a few replication forks are arrested under normal growth conditions. This assumption, however, is at odds with the severe and deleterious phenotypes associated with a null mutant of priA, the gene encoding a helicase implicated in the reactivation of arrested replication forks. We developed an assay that involves an abrupt inactivation of rounds of synchronized replication in a large population of cells, in order to evaluate the ability of dnaC2 cells to reactivate arrested replication forks at non-permissive temperature. We compared the rate at which arrested replication forks accumulated in dnaC2 priA+ and dnaC2 priA2 cells and observed that this rate was lower in dnaC2 priA+ cells. We conclude that while replication cannot initiate in a dnaC2 mutant at non-permissive temperature, a class of arrested replication forks (PriA-dependent and DnaC-independent) are reactivated within these cells.

A Single Residue Determines the Cooperative Binding Property of a Primosomal DNA Replication Protein, PriB, to Single-Stranded DNA

PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)25, and (StPriB)25 are proposed.

The priB Gene of Klebsiella pneumoniae Encodes a 104-Amino Acid Protein That Is Similar in Structure and Function to Escherichia coli PriB

Primosome protein PriB is a single-stranded DNA-binding protein that serves as an accessory factor for PriA helicase-catalyzed origin-independent reinitiation of DNA replication in bacteria. A recent report describes the identification of a novel PriB protein in Klebsiella pneumoniae that is significantly shorter than most sequenced PriB homologs. The K. pneumoniae PriB protein is proposed to comprise 55 amino acid residues, in contrast to E. coli PriB which comprises 104 amino acid residues and has a length that is typical of most sequenced PriB homologs. Here, we report results of a sequence analysis that suggests that the priB gene of K. pneumoniae encodes a 104-amino acid PriB protein, akin to its E. coli counterpart. Furthermore, we have cloned the K. pneumoniae priB gene and purified the 104-amino acid K. pneumoniae PriB protein. Gel filtration experiments reveal that the K. pneumoniae PriB protein is a dimer, and equilibrium DNA binding experiments demonstrate that K. pneumoniae PriB's single-stranded DNA-binding activity is similar to that of E. coli PriB. These results indicate that the PriB homolog of K. pneumoniae is similar in structure and in function to that of E. coli.

A bacterial PriB with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase

BackgroundBacterial DNA replication restart pathways facilitate reinitiation of DNA replication following disruptive encounters of a replisome with DNA damage, thereby allowing complete and faithful duplication of the genome. In Neisseria gonorrhoeae, the primosome proteins that catalyze DNA replication restart differ from the well-studied primosome proteins of E. coli with respect to the number of proteins involved and the affinities of their physical interactions: the PriA:PriB interaction is weak in E. coli, but strong in N. gonorrhoeae, and the PriB:DNA interaction is strong in E. coli, but weak in N. gonorrhoeae. In this study, we investigated the functional consequences of this affinity reversal.ResultsWe report that N. gonorrhoeae PriA's DNA binding and unwinding activities are similar to those of E. coli PriA, and N. gonorrhoeae PriA's helicase activity is stimulated by its cognate PriB, as it is in E. coli. This finding is significant because N. gonorrhoeae PriB's single-stranded DNA binding activity is weak relative to that of E. coli PriB, and in E. coli, PriB's single-stranded DNA binding activity is important for PriB stimulation of PriA helicase. Furthermore, a N. gonorrhoeae PriB variant defective for binding single-stranded DNA can stimulate PriA's helicase activity, suggesting that DNA binding by PriB might not be important for PriB stimulation of PriA helicase in N. gonorrhoeae. We also demonstrate that N. gonorrhoeae PriB stimulates ATP hydrolysis catalyzed by its cognate PriA. This activity of PriB has not been observed in E. coli, and could be important for PriB stimulation of PriA helicase in N. gonorrhoeae.ConclusionsThe results of this study demonstrate that a bacterial PriB homolog with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase. While it remains unclear if N. gonorrhoeae PriB's weak DNA binding activity is required for PriB stimulation of PriA helicase, the ability of PriB to stimulate PriA-catalyzed ATP hydrolysis could play an important role. Thus, the weak interaction between N. gonorrhoeae PriB and DNA might be compensated for by the strong interaction between PriB and PriA, which could result in allosteric activation of PriA's ATPase activity.

Oligonucleotides stimulate genomic alterations of Legionella pneumophila

Genetic variation generates diversity in all kingdoms of life. The corresponding mechanisms can also be harnessed for laboratory studies of fundamental cellular processes. Here we report that oligonucleotides (oligos) generate mutations on the Legionella pneumophila chromosome by a mechanism that requires homologous DNA, but not RecA, RadA or any known phage recombinase. Instead we propose that DNA replication contributes, as oligo-induced mutagenesis required ≥ 21 nucleotides of homology, was strand-dependent, and was most efficient in exponential phase. Mutagenesis did not require canonical 5' phosphate or 3' hydroxyl groups, but the primosomal protein PriA and DNA Pol I contributed. After electroporation, oligos stimulated excision of 2.1 kb of chromosomal DNA or insertion of 18 bp, and non-homologous flanking sequences were also processed. We exploited this endogenous activity to generate chromosomal deletions and to insert an epitope into a chromosomal coding sequence. Compared with Escherichia coli, L. pneumophila encodes fewer canonical single-stranded exonucleases, and the frequency of mutagenesis increased substantially when either its RecJ and ExoVII nucleases were inactivated or the oligos modified by nuclease-resistant bases. In addition to genetic engineering, oligo-induced mutagenesis may have evolutionary implications as a mechanism to incorporate divergent DNA sequences with only short regions of homology.

Identification of a novel protein, PriB, in Klebsiella pneumoniae

PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L45, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed.

Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives.

The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies.

Primosomal Proteins DnaD and DnaB Are Recruited to Chromosomal Regions Bound by DnaA inBacillus subtilis

The initiation of DNA replication requires the binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. DnaA also binds to many sites around the chromosome, outside oriC, and acts as a transcription factor at several of these. In low-G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase at oriC, and this depends on DnaA. DnaD and DnaB also are required to load the replicative helicase outside oriC during replication restart, independently of DnaA. Using chromatin immunoprecipitation, we found that DnaD and DnaB, but not the replicative helicase, are associated with many of the chromosomal regions bound by DnaA in Bacillus subtilis. This association was dependent on DnaA, and the order of recruitment was the same as that at oriC, but it was independent of a functional oriC and suggests that DnaD and DnaB do not require open complex formation for the stable association with DNA. These secondary binding regions for DnaA could be serving as a reservoir for excess DnaA, DnaD, and DnaB to help properly regulate replication initiation and perhaps are analogous to the proposed function of the datA locus in Escherichia coli. Alternatively, DnaD and DnaB might modulate the activity of DnaA at the secondary binding regions. All three of these proteins are widely conserved and likely have similar functions in a range of organisms.

Enterovirus 3C Proteases: Structure-based Discovery of Inhibitors

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.