title: The helicase-binding domain of Escherichia coli DnaG primase interacts with the highly conserved C-terminal region of single-stranded DNA-binding protein.
abstract: During bacterial DNA replication, DnaG primase and the χ subunit of DNA polymerase III compete for binding to single-stranded DNA-binding protein (SSB), thus facilitating the switch between priming and elongation. SSB proteins play an essential role in DNA metabolism by protecting single-stranded DNA and by mediating several important protein-protein interactions. Although an interaction of SSB with primase has been previously reported, it was unclear which domains of the two proteins are involved. This study identifies the C-terminal helicase-binding domain of DnaG primase (DnaG-C) and the highly conserved C-terminal region of SSB as interaction sites. By ConSurf analysis, it can be shown that an array of conserved amino acids on DnaG-C forms a hydrophobic pocket surrounded by basic residues, reminiscent of known SSB-binding sites on other proteins. Using protein-protein cross-linking, site-directed mutagenesis, analytical ultracentrifugation and nuclear magnetic resonance spectroscopy, we demonstrate that these conserved amino acid residues are involved in the interaction with SSB. Even though the C-terminal domain of DnaG primase also participates in the interaction with DnaB helicase, the respective binding sites on the surface of DnaG-C do not overlap, as SSB binds to the N-terminal subdomain, whereas DnaB interacts with the ultimate C-terminus.
authors: Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors: Natalie
authors: N
authors: Naue
authors: Monika
authors: M
authors: Beerbaum
authors: Andrea
authors: A
authors: Bogutzki
authors: Peter
authors: P
authors: Schmieder
authors: Ute
authors: U
authors: Curth
mesh_terms: D001665:Binding Sites; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010455:Peptides; C476430:SSB protein, E coli; D019915:DNA Primase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C121791:dnaG protein, E coli; D008239:Lysine