Site-directed mutagenesis of the χ subunit of DNA polymerase III and single-stranded DNA-binding protein of E. coli reveals key residues for their interaction.
During DNA replication in Escherichia coli, single-stranded DNA-binding protein (SSB) protects single-stranded DNA from nuclease action and hairpin formation. It is known that the highly conserved C-terminus of SSB contacts the χ subunit of DNA polymerase III. However, there only exists a theoretical model in which the 11 C-terminal amino acids of SSB have been docked onto the surface of χ. In order to refine this model of SSB/χ interaction, we exchanged amino acids in χ and SSB by site-directed mutagenesis that are predicted to be of key importance. Detailed characterization of the interaction of these mutants by analytical ultracentrifugation shows that the interaction area is correctly predicted by the model; however, the SSB C-terminus binds in a different orientation to the χ surface. We show that evolutionary conserved residues of χ form a hydrophobic pocket to accommodate the ultimate two amino acids of SSB, P176 and F177. This pocket is surrounded by conserved basic residues, important for the SSB/χ interaction. Mass spectrometric analysis of χ protein cross-linked to a C-terminal peptide of SSB reveals that K132 of χ and D172 of SSB are in close contact. The proposed SSB-binding site resembles those described for RecQ and exonuclease I.
Naue
Natalie
N
Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
Fedorov
Roman
R
Pich
Andreas
A
Manstein
Dietmar J
DJ
Curth
Ute
U
D001665:Binding Sites; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D014443:Tyrosine / Q000737:chemistry
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli; D014443:Tyrosine; D010649:Phenylalanine; D004258:DNA Polymerase III; C476431:holC protein, E coli; D008239:Lysine