Limits...
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.

Naue N, Fedorov R, Pich A, Manstein DJ, Curth U - Nucleic Acids Res. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Interaction of different mutants of EcoSSB and χ under high salt conditions. An amount of 2.5 µM of EcoSSB was mixed with different amounts of χ in a buffer containing 20 mM potassium phosphate pH 7.4, 0.3 M NaCl and 0.5 mM DTT and analysed in sedimentation velocity experiments in an analytical ultracentrifuge. In A, B and D, a binding isotherm fitted to the data of the interaction of EcoSSB and χ wild-type (red triangle) with KA = (2.9 ± 1) × 105 M−1 and n = 4.2 is shown for comparison (solid red line). (A) Exchanging V117 to phenylalanine (inverted blue triangle) dramatically lowered the binding affinity between χ and EcoSSB. Also an extension of SSB by a C-terminal glycine (SSB+Gly) disabled interaction with χ (red rhombus). Replacing Y119 by alanine (blue square) did not significantly change the affinity of χ to SSB [dashed blue line: KA = (3.4 ± 1) × 105 M−1 and n = 4.2]. (B) Exchanging tyrosine 131 of χ to leucine dramatically lowered the binding affinity to SSB (red square), whereas χ Y131A retained some of its activity (blue rhombus). The dashed blue line represents a binding isotherm fitted to the data of χ Y131A binding to EcoSSB with KA = (4.8 ± 1) × 104 M−1, n = 4.2. (C) The K124A mutation had a strong effect on the SSB/χ interaction (red square), lowering KA to (2.1 ± 0.5) × 104 M−1 with n = 4.2 (solid red line). The χ K132A mutant (blue triangle) also displayed a lowered binding affinity to SSB [dashed blue line: KA = (9 ± 2.5) × 104 M−1 and n = 4.2]. χ K132M (red rhombus) behaved similar to wild-type protein [fine dashed red line: KA = (2.1 ± 1) × 105 M−1 and n = 4.2]. (D) In order to test the new model of SSB/χ interaction, the K124M and R128A mutants of χ were generated. Both showed effects in accordance with the new model, with K124M (blue rhombus) lowering the binding affinity to SSB to a fifth compared to wild-type [dashed blue line, KA = (6 ± 1) × 104 M−1 and n = 4.2] and R128A (red squares) disabling the interaction with SSB.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045619&req=5

Figure 1: Interaction of different mutants of EcoSSB and χ under high salt conditions. An amount of 2.5 µM of EcoSSB was mixed with different amounts of χ in a buffer containing 20 mM potassium phosphate pH 7.4, 0.3 M NaCl and 0.5 mM DTT and analysed in sedimentation velocity experiments in an analytical ultracentrifuge. In A, B and D, a binding isotherm fitted to the data of the interaction of EcoSSB and χ wild-type (red triangle) with KA = (2.9 ± 1) × 105 M−1 and n = 4.2 is shown for comparison (solid red line). (A) Exchanging V117 to phenylalanine (inverted blue triangle) dramatically lowered the binding affinity between χ and EcoSSB. Also an extension of SSB by a C-terminal glycine (SSB+Gly) disabled interaction with χ (red rhombus). Replacing Y119 by alanine (blue square) did not significantly change the affinity of χ to SSB [dashed blue line: KA = (3.4 ± 1) × 105 M−1 and n = 4.2]. (B) Exchanging tyrosine 131 of χ to leucine dramatically lowered the binding affinity to SSB (red square), whereas χ Y131A retained some of its activity (blue rhombus). The dashed blue line represents a binding isotherm fitted to the data of χ Y131A binding to EcoSSB with KA = (4.8 ± 1) × 104 M−1, n = 4.2. (C) The K124A mutation had a strong effect on the SSB/χ interaction (red square), lowering KA to (2.1 ± 0.5) × 104 M−1 with n = 4.2 (solid red line). The χ K132A mutant (blue triangle) also displayed a lowered binding affinity to SSB [dashed blue line: KA = (9 ± 2.5) × 104 M−1 and n = 4.2]. χ K132M (red rhombus) behaved similar to wild-type protein [fine dashed red line: KA = (2.1 ± 1) × 105 M−1 and n = 4.2]. (D) In order to test the new model of SSB/χ interaction, the K124M and R128A mutants of χ were generated. Both showed effects in accordance with the new model, with K124M (blue rhombus) lowering the binding affinity to SSB to a fifth compared to wild-type [dashed blue line, KA = (6 ± 1) × 104 M−1 and n = 4.2] and R128A (red squares) disabling the interaction with SSB.

Mentions: The amount of bound χ per EcoSSB was plotted against the amount of total χ per EcoSSB and a binding isotherm for independent binding of n χ molecules to one EcoSSB molecule was fitted to the data (Figure 1A) (25). The resulting binding isotherm shows that one EcoSSB tetramer can bind up to four molecules of χ with an affinity (KA) of about 3 × 105 M−1 (Figure 1A). These results are in good agreement with previously published data (25,50). An overview of all binding constants determined in this study can be found in Table 1.Figure 1.


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.

Naue N, Fedorov R, Pich A, Manstein DJ, Curth U - Nucleic Acids Res. (2010)

Interaction of different mutants of EcoSSB and χ under high salt conditions. An amount of 2.5 µM of EcoSSB was mixed with different amounts of χ in a buffer containing 20 mM potassium phosphate pH 7.4, 0.3 M NaCl and 0.5 mM DTT and analysed in sedimentation velocity experiments in an analytical ultracentrifuge. In A, B and D, a binding isotherm fitted to the data of the interaction of EcoSSB and χ wild-type (red triangle) with KA = (2.9 ± 1) × 105 M−1 and n = 4.2 is shown for comparison (solid red line). (A) Exchanging V117 to phenylalanine (inverted blue triangle) dramatically lowered the binding affinity between χ and EcoSSB. Also an extension of SSB by a C-terminal glycine (SSB+Gly) disabled interaction with χ (red rhombus). Replacing Y119 by alanine (blue square) did not significantly change the affinity of χ to SSB [dashed blue line: KA = (3.4 ± 1) × 105 M−1 and n = 4.2]. (B) Exchanging tyrosine 131 of χ to leucine dramatically lowered the binding affinity to SSB (red square), whereas χ Y131A retained some of its activity (blue rhombus). The dashed blue line represents a binding isotherm fitted to the data of χ Y131A binding to EcoSSB with KA = (4.8 ± 1) × 104 M−1, n = 4.2. (C) The K124A mutation had a strong effect on the SSB/χ interaction (red square), lowering KA to (2.1 ± 0.5) × 104 M−1 with n = 4.2 (solid red line). The χ K132A mutant (blue triangle) also displayed a lowered binding affinity to SSB [dashed blue line: KA = (9 ± 2.5) × 104 M−1 and n = 4.2]. χ K132M (red rhombus) behaved similar to wild-type protein [fine dashed red line: KA = (2.1 ± 1) × 105 M−1 and n = 4.2]. (D) In order to test the new model of SSB/χ interaction, the K124M and R128A mutants of χ were generated. Both showed effects in accordance with the new model, with K124M (blue rhombus) lowering the binding affinity to SSB to a fifth compared to wild-type [dashed blue line, KA = (6 ± 1) × 104 M−1 and n = 4.2] and R128A (red squares) disabling the interaction with SSB.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045619&req=5

Figure 1: Interaction of different mutants of EcoSSB and χ under high salt conditions. An amount of 2.5 µM of EcoSSB was mixed with different amounts of χ in a buffer containing 20 mM potassium phosphate pH 7.4, 0.3 M NaCl and 0.5 mM DTT and analysed in sedimentation velocity experiments in an analytical ultracentrifuge. In A, B and D, a binding isotherm fitted to the data of the interaction of EcoSSB and χ wild-type (red triangle) with KA = (2.9 ± 1) × 105 M−1 and n = 4.2 is shown for comparison (solid red line). (A) Exchanging V117 to phenylalanine (inverted blue triangle) dramatically lowered the binding affinity between χ and EcoSSB. Also an extension of SSB by a C-terminal glycine (SSB+Gly) disabled interaction with χ (red rhombus). Replacing Y119 by alanine (blue square) did not significantly change the affinity of χ to SSB [dashed blue line: KA = (3.4 ± 1) × 105 M−1 and n = 4.2]. (B) Exchanging tyrosine 131 of χ to leucine dramatically lowered the binding affinity to SSB (red square), whereas χ Y131A retained some of its activity (blue rhombus). The dashed blue line represents a binding isotherm fitted to the data of χ Y131A binding to EcoSSB with KA = (4.8 ± 1) × 104 M−1, n = 4.2. (C) The K124A mutation had a strong effect on the SSB/χ interaction (red square), lowering KA to (2.1 ± 0.5) × 104 M−1 with n = 4.2 (solid red line). The χ K132A mutant (blue triangle) also displayed a lowered binding affinity to SSB [dashed blue line: KA = (9 ± 2.5) × 104 M−1 and n = 4.2]. χ K132M (red rhombus) behaved similar to wild-type protein [fine dashed red line: KA = (2.1 ± 1) × 105 M−1 and n = 4.2]. (D) In order to test the new model of SSB/χ interaction, the K124M and R128A mutants of χ were generated. Both showed effects in accordance with the new model, with K124M (blue rhombus) lowering the binding affinity to SSB to a fifth compared to wild-type [dashed blue line, KA = (6 ± 1) × 104 M−1 and n = 4.2] and R128A (red squares) disabling the interaction with SSB.
Mentions: The amount of bound χ per EcoSSB was plotted against the amount of total χ per EcoSSB and a binding isotherm for independent binding of n χ molecules to one EcoSSB molecule was fitted to the data (Figure 1A) (25). The resulting binding isotherm shows that one EcoSSB tetramer can bind up to four molecules of χ with an affinity (KA) of about 3 × 105 M−1 (Figure 1A). These results are in good agreement with previously published data (25,50). An overview of all binding constants determined in this study can be found in Table 1.Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus