Structural insight into the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.
ABSTRACT: 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.
Project description: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.
Project description: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.
Project description:BACKGROUND: Bacterial 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. RESULTS: We 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. CONCLUSIONS: The 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.
Project description:Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 A resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.
Project description: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 ?-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 ?-hairpin eliminated PriA DNA unwinding, interaction with the primosome protein PriB, and cellular function. We isolated a spontaneous intragenic suppressor mutant of the priA ?-hairpin deletion mutant in which 22 codons around the deletion site were duplicated. This suppressor variant and an Ala-substituted ?-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.
Project description:Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved 'DNA replication restart' pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT).
Project description:The ability to restart broken DNA replication forks is essential across all domains of life. In Escherichia coli, the priA, priB, priC, and dnaT genes encode the replication restart proteins (RRPs) to accomplish this task. PriA plays a critical role in replication restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par-), UV sensitivity, and recombination deficiency (Rec-). PriA has 733 amino acids, and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, remodel the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB, and DnaT, and display ATPase, helicase, and translocase activities. We have characterized a new priA mutation called priA316::cat It is a composite mutation involving an insertion that truncates the protein within the winged-helix domain (at the 154th codon) and an ACG (Thr)-to-ATG (Met) mutation that allows reinitiation of translation at the 157th codon such that PriA is expressed in two pieces. priA316::cat phenotypes are like those of the wild type for growth, recombination, and UV resistance, revealing only a slightly increased level of SOS expression and defects in nucleoid partitioning in the mutant. Both parts of PriA are required for activity, and the N-terminal fragment can be optimized to yield wild-type activity. A deletion of the lon protease suppresses priA316::cat phenotypes. We hypothesize the two parts of PriA form a complex that supplies most of the PriA activity needed in the cell.IMPORTANCE PriA is a highly conserved multifunctional protein that plays a crucial role in the essential process of replication restart. Here we characterize an insertion mutation of priA with an intragenic suppressor such that it is now made in two parts. These two pieces split the winged-helix domain to separate the N-terminal 3' DNA-binding domain from the C-terminal domain of PriA. It is hypothesized that the two pieces form a complex that is capable of almost wild type priA function. The composite mutation leads to a moderate level of SOS expression and defects in partitioning of the chromosomes. Full function is restored by deletion of lon, suggesting that stability of this complex may be a reason for the partial phenotypes seen.
Project description: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.
Project description:PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 A resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB-dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB-ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L45 plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA-PriB-ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.
Project description: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.