ABSTRACT
Reactivation of repaired DNA replication forks in bacteria is catalyzed by PriA helicase. This broadly-conserved bacterial enzyme can remodel the structure of DNA at a repaired DNA replication fork by unwinding small portions of duplex DNA to prepare the fork for replisome reloading. While PriA's helicase activity is not strictly required for cell viability in E. coli, the sequence motifs that confer helicase activity upon PriA are well-conserved among sequenced bacterial priA genes, suggesting that PriA's duplex DNA unwinding activity confers a selective advantage upon cells. However, these helicase sequence motifs are not well-conserved among priA genes from the Deinococcus-Thermus phylum. Here, we show that PriA from a highly radiation-resistant member of that phylum, Deinococcus radiodurans, lacks the ability to hydrolyze ATP and unwind duplex DNA, thus qualifying D. radiodurans PriA as a pseudohelicase. Despite the lack of helicase activity, D. radiodurans PriA has retained the DNA binding activity expected of a typical PriA helicase, and we present evidence for a physical interaction between D. radiodurans PriA and its cognate replicative helicase, DnaB. This suggests that PriA has retained a role in replisome reloading onto repaired DNA replication forks in D. radiodurans despite its lack of helicase activity.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/physiology , DNA Helicases/genetics , DNA Helicases/physiology , Deinococcus/enzymology , Adenosine Triphosphate/chemistry , Amino Acid Motifs , Base Sequence , Catalysis , Cloning, Molecular , DNA/chemistry , DNA Repair , DNA Replication , Deinococcus/genetics , DnaB Helicases/genetics , Electrophoresis, Agar Gel , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/physiology , Evolution, Molecular , Guanidine/chemistry , Hydrolysis , Molecular Sequence Data , Mutation , Protein Denaturation , Sequence Homology, Nucleic AcidABSTRACT
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.
Subject(s)
DNA Helicases/antagonists & inhibitors , Enzyme Inhibitors/analysis , Escherichia coli Proteins/antagonists & inhibitors , Quinazolines/pharmacology , Triazoles/pharmacology , Adenosine Triphosphate/metabolism , Binding Sites , DNA Helicases/metabolism , DNA-Binding Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/metabolism , Inhibitory Concentration 50 , Kinetics , Neisseria gonorrhoeae/enzymology , Protein Conformation/drug effectsABSTRACT
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.
Subject(s)
DNA-Binding Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Klebsiella pneumoniae/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Chromatography, Gel , DNA-Binding Proteins/metabolism , Molecular Sequence Data , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Species Specificity , TemperatureABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , DNA Helicases/metabolism , DNA, Bacterial/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Neisseria gonorrhoeae/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA Helicases/chemistry , DNA Helicases/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Neisseria gonorrhoeae/chemistry , Neisseria gonorrhoeae/enzymology , Neisseria gonorrhoeae/genetics , Nucleic Acid Conformation , Protein BindingABSTRACT
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.
