DNA Replication Restart Research Articles

DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii.

Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea.

Inhibition of Staphylococcus aureus PriA Helicase by Flavonol Kaempferol.

Staphylococcus aureus is an important etiological agent responsible for healthcare-associated infections. In this study, the effect of flavonoids on the inhibition of S. aureus PriA (SaPriA), an essential helicase for DNA replication restart, which is critical for bacterial survival, was investigated. Using vanadate-sensitive colorimetric assay, the concentration of phosphate, from ATP hydrolysis by SaPriA, was decreased to 37 and 69 %, respectively, in the presence of 35 μM kaempferol and myricetin. The effect of quercetin, galangin, dihydromyricetin, and myricitrin was insignificant. From titration curve, IC50 of kaempferol for SaPriA was determined to be 22 ± 2 μM. Using fluorescence quenching, we identified that kaempferol can bind to SaPriA with K d of 9.1 ± 3.2 μM. To our knowledge, these preliminary results constituted the first study regarding that naturally occurring product such as flavonols kaempferol and myricetin can be potent inhibitors targeting PriA.

RAD50 phosphorylation promotes ATR downstream signaling and DNA restart following replication stress

The MRE11/RAD50/NBN (MRN) complex plays a key role in detecting DNA double-strand breaks, recruiting and activating ataxia-telangiectasia mutated and in processing the breaks. Members of this complex also act as adaptor molecules for downstream signaling to the cell cycle and other cellular processes. Somewhat more controversial are the results to support a role for MRN in the ataxia-telangiectasia and Rad3-related (ATR) activation and signaling. We provide evidence that RAD50 is required for ATR activation in mammalian cells in response to DNA replication stress. It is in turn phosphorylated at a specific site (S635) by ATR, which is required for ATR signaling through Chk1 and other downstream substrates. We find that RAD50 phosphorylation is essential for DNA replication restart by promoting loading of cohesin at these sites. We also demonstrate that replication stress-induced RAD50 phosphorylation is functionally significant for cell survival and cell cycle checkpoint activation. These results highlight the importance of the adaptor role for a member of the MRN complex in all aspects of the response to DNA replication stress.

Mechanistic insights into PriA mediated DNA replication restart (924.2)

DNA replication restart is an essential genome maintenance process by which replication complexes (replisomes) are reloaded onto abandoned DNA replication forks. In Escherichia coli and related bacteria this process is orchestrated by the PriA DNA helicase, which binds to replication forks in a structure‐specific manner. PriA also binds to single‐stranded DNA‐binding protein (SSB), a protein that coats the lagging‐strand template at DNA replication forks. Complex formation with SSB stimulates PriA DNA unwinding DNA and, as we show here, this interaction is required for PriA remodeling of SSB‐DNA nucleoprotein complexes. Proteins that interact with SSB typically do so by binding directly to the evolutionarily conserved amphipathic C‐terminal tail of SSB (SSB‐CT). Although previous studies indicated that PriA also binds to SSB via the SSB‐CT, a lack of structural information on PriA has left the SSB binding site on PriA unknown. We determined the 4 Å resolution structure of PriA bound to the SSB‐CT. The electrostatic characteristics of the PriA SSB‐Ct binding site resemble those of other known SSB‐interacting proteins. Single‐molecule FRET studies show that a PriA variant that has lost the ability to bind the SSB‐CT fails to modulate SSB‐DNA complexes. Our studies point to the importance of the PriA/SSB interaction in allowing PriA to reload replisomes at damaged replication forks where SSB is bound to the lagging‐strand template.

Structural mechanisms of DNA replication restart (352.2)

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to the completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved “DNA replication restart” mechanisms that reload replisomes onto abandoned DNA replication forks and D‐loop structures that result from recombinational DNA repair. In an early step in this process, the PriA DNA helicase binds to the fork/D‐loop structure in a structure‐specific (sequence‐independent) manner ‐ this DNA‐bound form of PriA then mediates assembly of the replication restart primosome, which reloads the replicative helicase (DnaB in E. coli). We have recently determined the X‐ray crystal structure of a bacterial PriA, which reveals a core helicase domain surrounded by auxiliary DNA‐binding domains that appear to be poised for specific binding of branched DNA structures. Mechanistic insights from this structure and others will be highlighted in my talk.

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.

Utilising Super-Resolution Palm Imaging in Fission Yeast

Super-resolution techniques such as PALM and STORM are an exciting development allowing researchers to break the diffraction limit imposed by the diffraction of light. Our group works on understanding the systems of DNA repair and replication restart using the model organism Schizosaccharomyces pombe (fission yeast). A recent paper by Lando et al., utilised PALM super-resolution imaging to count the number of a protein tagged with the photo-activatable protein mEos2 in fission yeast. The potential to determine the precise stoichiometry of protein complexes in vivo would be an important breakthrough. To do this it is necessary to establish the ideal protocols for super-resolution imaging in S. pombe, and what the limitations of this technique are. We present the results of work with Cdc22, a protein highly expressed throughout the cell in fission yeast, known to form hexamers, with a control mutant impaired in hexamer formation. The protein was labelled genomically with the photo-activatable protein mEos2 to allow it to be visualised. We have established optimal excitation power and temporal regimes to visualise Cdc22 in S. pombe, as well as protocols to significantly reduce intracellular background fluorescence.

Structural mechanisms of PriA-mediated DNA replication restart

Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.

PriC-mediated DNA Replication Restart Requires PriC Complex Formation with the Single-stranded DNA-binding Protein

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D

BackgroundStalled replication forks at common fragile sites are a major cause of genomic instability. RecQ helicases, a highly conserved family of DNA-unwinding enzymes, are believed to ease ‘roadblocks’ that pose challenge to replication fork progression. Among the five known RecQ homologs in humans, functions of RECQ1, the most abundant of all, are poorly understood. We previously determined that RECQ1 helicase preferentially binds and unwinds substrates that mimic DNA replication/repair intermediates, and interacts with proteins involved in DNA replication restart mechanisms.MethodWe have utilized chromatin immunoprecipitation followed by quantitative real-time PCR to investigate chromatin interactions of RECQ1 at defined genetic loci in the presence or absence of replication stress. We have also tested the sensitivity of RECQ1-depleted cells to aphidicolin induced replication stress.ResultsRECQ1 binds to the origins of replication in unperturbed cells. We now show that conditions of replication stress induce increased accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, RECQ1 is specifically enriched at two major fragile sites FRA3B and FRA16D where replication forks have stalled following aphidicolin treatment. RECQ1-depletion results in attenuated checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability.ConclusionsGiven a recent biochemical observation that RECQ1 catalyzes strand exchange on stalled replication fork structures in vitro, our results indicate that RECQ1 facilitates repair of stalled or collapsed replication forks and preserves genome integrity. Our findings provide the first evidence of a crucial role for RECQ1 at naturally occurring fork stalling sites and implicate RECQ1 in mechanisms underlying common fragile site instability in cancer.

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 PriA Replication Restart Protein Blocks Replicase Access Prior to Helicase Assembly and Directs Template Specificity through Its ATPase Activity

The PriA protein serves as an initiator for the restart of DNA replication on stalled replication forks and as a checkpoint protein that prevents the replicase from advancing in a strand displacement reaction on forks that do not contain a functional replicative helicase. We have developed a primosomal protein-dependent fluorescence resonance energy transfer (FRET) assay using a minimal fork substrate composed of synthetic oligonucleotides. We demonstrate that a self-loading reaction, which proceeds at high helicase concentrations, occurs by threading of a preassembled helicase over free 5'-ends, an event that can be blocked by attaching a steric block to the 5'-end or coating DNA with single-stranded DNA binding protein. The specificity of PriA for replication forks is regulated by its intrinsic ATPase. ATPase-defective PriA K230R shows a strong preference for substrates that contain no gap between the leading strand and the duplex portion of the fork, as demonstrated previously. Wild-type PriA prefers substrates with larger gaps, showing maximal activity on substrates on which PriA K230R is inactive. We demonstrate that PriA blocks replicase function on forks by blocking its binding.

Identification of a Small Molecule PriA Helicase Inhibitor

PriA helicase catalyzes the initial steps of replisome reloading onto repaired DNA replication forks in bacterial DNA replication restart pathways. We have used a high-throughput screen to identify a small molecule inhibitor of PriA-catalyzed duplex DNA unwinding. The compound, CGS 15943, targets Neisseria gonorrhoeae PriA helicase with an IC(50) of 114 ± 24 μM. The PriA helicase of Escherichia coli is also inhibited, although to a lesser extent than N. gonorrhoeae PriA. CGS 15943 decreases rates of PriA-catalyzed ATP hydrolysis and reduces the affinity with which PriA binds DNA. Steady-state kinetic data indicate that CGS 15943 inhibits PriA through a mixed mode of inhibition with respect to ATP and with respect to DNA, indicating that it binds to a site on PriA that participates in both substrate binding and catalysis. Inhibitor binding constants derived from steady-state kinetic experiments reveal that CGS 15943 has the highest binding affinity for the PriA·PriB·ATP complex, intermediate binding affinity for the PriA·PriB·DNA complex, and the lowest binding affinity for the PriA·PriB·DNA·ATP complex, suggesting that PriA assumes different conformations in each of these complexes. We propose that CGS 15943 binds to PriA at a site distinct from the DNA and primary ATP binding sites, perhaps at PriA's weak nucleotide binding site, and induces a conformational change in PriA that renders it less catalytically proficient or prevents conformational changes in PriA that are necessary for ATP hydrolysis and duplex DNA unwinding.

Binding Mechanism of Metal⋅NTP Substrates and Stringent-Response Alarmones to Bacterial DnaG-Type Primases

Primases are DNA-dependent RNA polymerases found in all cellular organisms. In bacteria, primer synthesis is carried out by DnaG, an essential enzyme that serves as a key component of DNA replication initiation, progression, and restart. How DnaG associates with nucleotide substrates and how certain naturally prevalent nucleotide analogs impair DnaG function are unknown. We have examined one of the earliest stages in primer synthesis and its control by solving crystal structures of the S.aureus DnaG catalytic core bound to metal ion cofactors and either individual nucleoside triphosphates or the nucleotidyl alarmones, pppGpp and ppGpp. These structures, together with both biochemical analyses and comparative studies of enzymes that use the same catalytic fold as DnaG, pinpoint the predominant nucleotide-binding site of DnaG and explain how the induction of the stringent response in bacteria interferes with primer synthesis.

Molecular Interactions in PriC‐mediated DNA Replication Restart

DNA replication restart is an essential process in bacteria that reloads DNA replication complexes (replisomes) that have ejected prematurely from replication forks due to encounters with impassable DNA damage or protein complexes. In E. coli and related bacterial species, the “replication restart proteins” (PriA, PriB, PriC, DnaT, and Rep) catalyze this activity. The 20‐kDa PriC protein is unique among these proteins in that it can mediate in vitro replisome reloading in the absence of other replication restart proteins. Precisely how PriC recognizes abandoned DNA replication fork structures and directs replisome reloading is currently unknown. We have found that the single‐strand DNA‐binding protein (SSB) forms a direct complex with PriC that we hypothesize is important for directing PriC reloading activities to abandoned DNA replication forks. PriC variants that fail that to bind SSB (but that retain DNA binding) were identified to test this hypothesis. Consistent with an essential role for the PriC/SSB interaction, these variants cannot catalyze the first step of replication restart (loading the DnaB helicase onto an SSB‐bound synthetic replication fork) in vitro. On‐going experiments aim to determine the solution structure of PriC and to assess the importance of complex formation with SSB in PriC‐mediated DNA replication restart in vivo. Research supported by GM068061 and T32‐GM07215.

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.

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.

Rtt107 Is Required for Recruitment of the SMC5/6 Complex to DNA Double Strand Breaks

Genome integrity is maintained by a network of DNA damage response pathways, including checkpoints and DNA repair processes. In Saccharomyces cerevisiae, the BRCT domain-containing protein Rtt107/Esc4 is required for the restart of DNA replication after successful repair of DNA damage and for cellular resistance to DNA-damaging agents. In addition to its well characterized interaction with the endonuclease Slx4, Rtt107 interacts with a number of other DNA repair and recombination proteins. These include the evolutionarily conserved SMC5/6 complex, which is involved in numerous chromosome maintenance activities, such as DNA repair, chromosome segregation, and telomere function. The interaction between Rtt107 and the SMC5/6 complex was mediated through the N-terminal BRCT domains of Rtt107 and the Nse6 subunit of SMC5/6 and was independent of methyl methane sulfonate-induced damage and Slx4. Supporting a shared function in the DNA damage response, Rtt107 was required for recruitment of SMC5/6 to DNA double strand breaks. However, this functional relationship did not extend to other types of DNA lesions such as protein-bound nicks. Interestingly, Rtt107 was phosphorylated when SMC5/6 function was compromised in the absence of DNA-damaging agents, indicating a connection beyond the DNA damage response. Genetic analyses revealed that, although a subset of Rtt107 and SMC5/6 functions was shared, these proteins also contributed independently to maintenance of genome integrity.

Loss of H3 K79 Trimethylation Leads to Suppression of Rtt107-dependent DNA Damage Sensitivity through the Translesion Synthesis Pathway

Genomic integrity is maintained by the coordinated interaction of many DNA damage response pathways, including checkpoints, DNA repair processes, and cell cycle restart. In Saccharomyces cerevisiae, the BRCA1 C-terminal domain-containing protein Rtt107/Esc4 is required for restart of DNA replication after successful repair of DNA damage and for cellular resistance to DNA-damaging agents. Rtt107 and its interaction partner Slx4 are phosphorylated during the initial phase of DNA damage response by the checkpoint kinases Mec1 and Tel1. Because the natural chromatin template plays an important role during the DNA damage response, we tested whether chromatin modifications affected the requirement for Rtt107 and Slx4 during DNA damage repair. Here, we report that the sensitivity to DNA-damaging agents of rtt107Δ and slx4Δ mutants was rescued by inactivation of the chromatin regulatory pathway leading to H3 K79 trimethylation. Further analysis revealed that lack of Dot1, the H3 K79 methyltransferase, led to activation of the translesion synthesis pathway, thereby allowing the survival in the presence of DNA damage. The DNA damage-induced phosphorylation of Rtt107 and Slx4, which was mutually dependent, was not restored in the absence of Dot1. The antagonistic relationship between Rtt107 and Dot1 was specific for DNA damage-induced phenotypes, whereas the genomic instability caused by loss of Rtt107 was not rescued. These data revealed a multifaceted functional relationship between Rtt107 and Dot1 in the DNA damage response and maintenance of genome integrity.

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.