Characterization of Staphylococcus aureus Primosomal DnaD Protein: Highly Conserved C-Terminal Region Is Crucial for ssDNA and PriA Helicase Binding but Not for DnaA Protein-Binding and Self-Tetramerization

Abstract

The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD) and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204) showed a stable tetramer in solution. This finding indicates that the C-terminal region aa 196–228 is not crucial for SaDnaD oligomerization. SaDnaD forms distinct complexes with ssDNA of different lengths. In fluorescence titrations, SaDnaD bound to ssDNA with a binding-site size of approximately 32 nt. A stable complex of SaDnaD1-195, SaDnaD1-200, and SaDnaD1-204 with ssDNA dT40 was undetectable, indicating that the C-terminal region of SaDnaD (particularly aa 205–228) is crucial for ssDNA binding. The SPR results revealed that SaDnaD1-195 can interact with SaDnaA but not with SaPriA, which may indicate that DnaD has different binding sites for PriA and DnaA. Both SaDnaD and SaDnaDY176A mutant proteins, but not SaDnaD1-195, can significantly stimulate the ATPase activity of SaPriA. Hence, the stimulation effect mainly resulted from direct contact within the protein—protein interaction, not via the DNA—protein interaction. Kinetic studies revealed that the SaDnaD-SaPriA interaction increases the Vmax of the SaPriA ATPase fivefold without significantly affecting the Km. These results indicate that the conserved C-terminal region is crucial for ssDNA and PriA helicase binding, but not for DnaA protein-binding and self-tetramerization.

The glycine-rich flexible region in SSB is crucial for PriA stimulation

“…KpPriA ATPase assay using the protocol described previously for SaPriA [28][29][30] was performed with 0.4 mM [g-32 P] ATP and 0.0625 mM KpPriA in reaction buffer containing 40 mM Tris (pH 8.0), 10 mM NaCl, 2 mM DTT, 2.5 mM MgCl 2 , and 0.1 mM PS4/PS3-dT30 DNA substrate. Aliquots (5 mL) 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 quantied with a phosphorimager.…”

Characterization of single-stranded DNA-binding protein SsbB fromStaphylococcus aureus: SsbB cannot stimulate PriA helicase

“…Construction of plasmids for SaSsbA, SaSsbB, SaDnaD, and SaPriA expression SaSsbA, 25 SaDnaD, 26 and SaPriA 30 expression plasmids have been constructed in other studies. SAAV0835, the gene encoding a putative SaSsbB, was amplied through PCR by using the genomic DNA of S. aureus subsp.…”

“…SaPriA ATPase assay 25,26 was performed with 0.4 mM [g-32 P] ATP and 0.12 mM SaPriA in a reaction buffer containing 40 mM Tris (pH 8.0), 10 mM NaCl, 2 mM DTT, 2.5 mM MgCl 2 , and 0.1 mM PS4/PS3-dT30 DNA substrate. Aliquots (5 mL) 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 min.…”

“…26 The manner by which SaSsbA and SaSsbB participate in SaPriA-directed primosome assembly and in DNA replication restart remains unclear.…”

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

“…Construction of the SaDnaD [ 58 ], SaPriA [ 59 ], Klebsiella pneumoniae SSB (KpSSB) [ 60 ], and tag-free KpSSB [ 61 ] expression plasmids has been reported. The gene encoding SaSsbA (the accession number ACY10277) was amplified by PCR using the genomic DNA of S .…”