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

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

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

Gel filtration of PriB proteins from <i>K. pneumoniae</i> and <i>E. coli.</i>

<p>Equivalent amounts of (a) <i>K. pneumoniae</i> PriB, and (b) <i>E. coli</i> PriB, each at approximately 3.4 g/l, were individually resolved through a sephacryl S-100 size-exclusion chromatography column under identical experimental conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024494#s3" target="_blank">Materials and Methods</a>.</p

Multiple amino acid sequence alignment of PriB homologs.

<p>Amino acid sequences of <i>Klebsiella pneumoniae</i> PriB (GenBank ID:YP_001338213), <i>Pectobacterium carotovorum</i> PriB (GenBank ID:C6DE14), <i>Yersinia ruckeri</i> PriB (GenBank ID:ZP_04617249), <i>Salmonella enterica</i> PriB (GenBank ID:AAL23212), <i>Escherichia coli</i> PriB (GenBank ID:NP_418622), and <i>Neisseria gonorrhoeae</i> PriB (GenBank ID:YP_207725) were aligned using the program ClustalX <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024494#pone.0024494-Thompson1" target="_blank">[15]</a>. Amino acid residues that are identical in at least five of the six aligned PriB proteins are highlighted in blue.</p

Single-stranded DNA-binding activity of <i>K. pneumoniae</i> PriB.

<p>PriB protein was diluted serially and incubated with fluorescein-labeled 15-base (circles), 30-base (squares), or 45-base (triangles) ssDNA oligonucleotides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024494#s3" target="_blank">Materials and Methods</a>. Measurements are reported in triplicate and error bars represent one standard deviation of the mean.</p

DNA binding activity of PriA.

<p><i>D</i>. <i>radiodurans</i> PriA (closed circles) and <i>E</i>. <i>coli</i> PriA (open circles) were individually tested for their ability to bind to A) an 18-base ssDNA oligonucleotide, or B) a fully-duplex fork DNA with 25 bp arms using a fluorescence-anisotropy-based equilibrium DNA binding assay as described in Materials and Methods. Data are reported in triplicate and error bars represent one standard deviation of the mean.</p

PriA-catalyzed ATP hydrolysis.

<p><i>D</i>. <i>radiodurans</i> PriA (closed circles) and <i>E</i>. <i>coli</i> PriA (open circles) were individually tested for their ability to catalyze hydrolysis of ATP in the presence of a 36 base dT homopolymer as described in Materials and Methods. Data are reported in triplicate and error bars represent one standard deviation of the mean.</p

Guanidine-mediated unfolding of PriA.

<p><i>D</i>. <i>radiodurans</i> PriA was incubated with indicated concentrations of guanidine-HCl and PriA’s intrinsic fluorescence was measured as described in Materials and Methods. The inset shows the fluorescence emission spectrum for <i>D</i>. <i>radiodurans</i> PriA in the presence and absence of 6 M guanidine-HCl. The best fit curve for the plot of the fraction of PriA folded as a function of guanidine-HCl was generated by fitting the data to a four-parameter function [y = (a + b·x)/(1 + c·x + d·x²)] (CurveExpert 1.3). Data are reported for one representative experiment.</p

Evolutionary conservation of PriA helicase.

<p>The amino acid sequences of PriA helicases from 19 bacterial species representing 7 distinct phyla are aligned with one another and represented by tan bars. The amino terminus of each PriA sequence is oriented at left. Conserved helicase motifs are labeled with roman numerals and are shown as blue boxes, and the cysteine-rich regions (CRR) are shown in red. Note that most of the length variation among PriA helicases maps to the amino-terminal region which contains the DNA binding domain.</p

Evolutionary conservation of PriA helicase motifs.

<p>Sequence logos were generated for helicase motifs I and II (the Walker A and Walker B boxes, respectively) from a multiple sequence alignment of 100 PriA helicases using the Weblogo 3 sequence logo generator [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133419#pone.0133419.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133419#pone.0133419.ref028" target="_blank">28</a>]. Each PriA sequence represents a unique genus, and 18 phyla are represented. A multiple sequence alignment of 17 members of the Deinococcus-Thermus phylum appears below each sequence logo. These sequences show significant departure from the otherwise well-conserved helicase motifs.</p