Conserved SSB-Ct binding site of PriA is not present in SaPriA.

<p>(A) SSB-Ct (DDIPF) binding site in KpPriA revealed by the complexed crystal structure (PDB ID: 4NL8). The KpPriA SSB-Ct binding site includes Trp82, Tyr86, Lys370, Arg697, and Gln701. (B) The putative SSB-Ct binding sites in SaPriA structurally corresponding with those in KpPriA are Trp89, Thr92, V439, Glu767, and Leu771. Only Trp89 in SaPriA (Trp82 in KpPriA) is conserved. Arg697, the most important residue in KpPriA in altering the SSB₃₅/SSB₆₅ distribution, is Glu767 in SaPriA. (C) Mutational analysis of SaPriA. The ATPase assay for the mutant SaPriA E767R and SaPriA R434A proteins was performed with 0.4 mM [γ-32P] ATP, 0.12 μM of the protein, SaSsbA (10 μM), and 0.1 μM PS4/PS3-dT30 DNA substrate for 1 h. Aliquots (5 μL) were taken and spotted onto a polyethyleneimine cellulose thin-layer chromatography plate, which was subsequently developed in 0.5 M formic acid and 0.25 M LiCl for 30 m. Reaction products were visualized by autoradiography and quantified with a Phosphorimager.</p

Data collection and processing statistics.

<p>Data collection and processing statistics.</p

Primers used for construction of plasmids.

<p>Primers used for construction of plasmids.</p

Oligomeric state of purified SaSsbA in solution.

<p>Purified protein in Buffer B was applied to a Superdex 200 prep grade column equilibrated with the same buffer. The column was calibrated with proteins of known molecular masses: thyroglobulin (670 kDa), γ-globulin (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa). The corresponding peak shows the eluted SaSsbA.</p

<i>Staphylococcus aureus</i> single-stranded DNA-binding protein SsbA can bind but cannot stimulate PriA helicase

<div><p>Single-stranded DNA-binding protein (SSB) and PriA helicase play important roles in bacterial DNA replication restart process. The mechanism by which PriA helicase is bound and stimulated by SSB in <i>Escherichia coli</i> (Ec) has been established, but information on this process in Gram-positive bacteria are limited. We characterized the properties of SSB from <i>Staphylococcus aureus</i> (SaSsbA, a counterpart of EcSSB) and analyzed its interaction with SaPriA. The gel filtration chromatography analysis of purified SaSsbA showed a stable tetramer in solution. The crystal structure of SaSsbA determined at 1.82 Å resolution (PDB entry 5XGT) reveals that the classic oligonucleotide/oligosaccharide-binding folds are formed in the N-terminal DNA-binding domain, but the entire C-terminal domain is disordered. Unlike EcSSB, which can stimulate EcPriA via a physical interaction between EcPriA and the C-terminus of EcSSB (SSB-Ct), SaSsbA does not affect the activity of SaPriA. We also found that SaPriA can be bound by SaSsbA, but not by SaSsbA-Ct. Although no effect was found with SaSsbA, SaPriA can be significantly stimulated by the Gram-negative <i>Klebsiella pneumoniae</i> SSB (KpSSB). In addition, we found that the conserved SSB-Ct binding site of KpPriA (Trp82, Tyr86, Lys370, Arg697, and Gln701) is not present in SaPriA. Arg697 in KpPriA is known to play a critical role in altering the SSB₃₅/SSB₆₅ distribution, but this corresponding residue in SaPriA is Glu767 instead, which has an opposite charge to Arg. SaPriA E767R mutant was constructed and analyzed; however, it still cannot be stimulated by SaSsbA. Finally, we found that the conserved MDFDDDIPF motif in the Gram-negative bacterial SSB is DISDDDLPF in SaSsbA, i.e., F172 in EcSSB and F168 in KpSSB is S161 in SaSsbA, not F. When acting with SaSsbA S161F mutant, the activity of SaPriA was dramatically enhanced elevenfold. Overall, the conserved binding sites, both in EcPriA and EcSSB, are not present in SaPriA and SaSsbA, thereby no stimulation occurs. Our observations through structure-sequence comparison and mutational analyses indicate that the case of EcPriA-EcSSB is not applicable to SaPriA-SaSsbA because of inherent differences among the species.</p></div

SPR analysis.

<p>(A) The SaPriA–SaSsbA interaction. SaPriA was immobilized on Series S sensor chips CM5, and the binding experiments were carried out using a Biacore T200. SaSsbA (1000, 500, 250, 125, and 63 nM) was injected in duplicate over the immobilized protein for 120 s at a flow rate of 30 μL/min. The estimated <i>K</i>_d value was derived by fitting the association and dissociation signals with a 1:1 (Langmuir) model using the Biacore T200 Evaluation Software. (B) The SaPriA–SaSsbA-Ct interaction analyzed by SPR. (C) The SaPriA–KpSSB-Ct interaction analyzed by SPR.</p

S161 in SaSsbA is a switch for SaPriA stimulation.

<p>(A) Multiple amino acid sequence alignment of SSB-Ct from Ec, Kp, St, Pa, and Sa. FDDDIPF in the C-terminal domain of SSB is usually conserved, but is not for SaSsbA. (B) SaPriA ATPase assay was performed with 0.4 mM [γ-32P] ATP, 0.12 μM of SaPriA, and 0.1 μM PS4/PS3-dT30 DNA substrate for 1 h. To study the effect, the mutant protein was individually added into the assay solution. Aliquots (5 μL) were taken and spotted onto a polyethyleneimine cellulose thin-layer chromatography plate, which was subsequently developed in 0.5 M formic acid and 0.25 M LiCl for 30 m. Reaction products were visualized by autoradiography and quantified with a Phosphorimager. Reaction was carried out without SaPriA (lane 1) or with SaPriA alone (lane 2) as controls. SaPriA acted with 5 μM SaSsbA plus 5 μM SaSsbA S161F mutant (lane 3), 10 μM SaSsbA S161F (lane 4), and 10 μM SaSsbA S161F/delI160 double mutant (lane 5) as shown, respectively.</p

The ATPase activity of SaPriA did not change when acting with SaSsbA.

<p>SaPriA ATPase assay was performed with 0.4 mM [γ-32P] ATP, 0.12 μM of SaPriA, and 0.1 μM PS4/PS3-dT30 (or dT30) DNA substrate for 1 h. To study the effect, SaSsbA (10 μM), tag-free SaSsbA (10 μM), or SaDnaD (4 μM) was added into the assay solution. Higher concentration of tag-free SaSsbA (20 μM; denotes ++) was also used in this study. Aliquots (5 μL) were taken and spotted onto a polyethyleneimine cellulose thin-layer chromatography plate, which was subsequently developed in 0.5 M formic acid and 0.25 M LiCl for 30 m. Reaction products were visualized by autoradiography and quantified with a Phosphorimager.</p

Sequence analysis of SaPriA.

<p>An alignment consensus of 417 sequenced PriA homologs by ConSurf reveals the degree of variability at each position along the sequence. In general, amino acid residues in the C-terminal region of PriA are conserved.</p

The [Protein]₅₀ values of SaSsbA as analyzed by EMSA.

<p>The [Protein]₅₀ values of SaSsbA as analyzed by EMSA.</p