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Distinct double- and single-stranded DNA binding of E. coli replicative DNA polymerase III alpha subunit.

McCauley MJ, Shokri L, Sefcikova J, Venclovas C, Beuning PJ, Williams MC - ACS Chem. Biol. (2008)

Bottom Line: In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force.From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA.The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, Boston, Massachusetts, 02115, USA.

ABSTRACT
The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.

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Binding isotherms for full-length protein and selected α constructs to DNA. a) Binding to dsDNA is determined by measuring the change in the melting force for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars represent the standard error from four experiments. Fits are to binding isotherms described within the text (eq 2 and eq 3), for n = 1 (solid lines) and n = 2 (dotted lines). For n = 1, Kds = 28 ± 7 × 106 M−1 for full-length α, 3.5 ± 0.9 × 106 M−1 for α1−917. and 0.4 ± 0.1 × 106 M−1 for α1−835. b) Binding to ssDNA is determined by measuring the fraction of ssDNA stabilized by protein binding, as found from eq 1, for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars are determined from errors in the fits. Fitting this data to the binding isotherms of the text (eq 3 and eq 4) determines Kss = 22 ± 6 × 106 M−1 for full-length α and 10 ± 3 × 106 M−1 for α917−1160. These results are summarized in Table 1.
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fig8: Binding isotherms for full-length protein and selected α constructs to DNA. a) Binding to dsDNA is determined by measuring the change in the melting force for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars represent the standard error from four experiments. Fits are to binding isotherms described within the text (eq 2 and eq 3), for n = 1 (solid lines) and n = 2 (dotted lines). For n = 1, Kds = 28 ± 7 × 106 M−1 for full-length α, 3.5 ± 0.9 × 106 M−1 for α1−917. and 0.4 ± 0.1 × 106 M−1 for α1−835. b) Binding to ssDNA is determined by measuring the fraction of ssDNA stabilized by protein binding, as found from eq 1, for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars are determined from errors in the fits. Fitting this data to the binding isotherms of the text (eq 3 and eq 4) determines Kss = 22 ± 6 × 106 M−1 for full-length α and 10 ± 3 × 106 M−1 for α917−1160. These results are summarized in Table 1.

Mentions: Stabilization of the double helix is shown in a series of extension (solid lines) and relaxation (dotted lines) cycles at varying salt concentrations. a, b) dsDNA is extended (black), and the helical form is disrupted at 62.6 ± 1.0 pN (32). As Na+ concentration is decreased, this transition occurs at lower forces and greater hysteresis is observed as DNA is relaxed (purple). As full-length protein is added (to the low-salt solution), the force required to disrupt the double helix increases again (blue). Furthermore, after a constant extension experiment in which the extension is fixed for 30 min, significant binding to the single strand appears (green), but only for the full-length protein (panel a) and not for the N-terminal α1−917 (panel b). c) The DNA melting force varies with Na+ concentration (purple), while melting force in the presence of 100 nM α and 500 nM α1−917 was observed to be independent of salt concentration. Uncertainties are determined from averages over three experiments. The average melting force for DNA, over the full range of Na+ concentrations studied, is 65.2 ± 0.4 pN in the presence of α protein and 65.1 ± 0.6 pN for α1−917. Higher concentrations of the α1−917 construct were necessary because of weaker binding (see Figure 5, panel b and Figure 8, panel a).


Distinct double- and single-stranded DNA binding of E. coli replicative DNA polymerase III alpha subunit.

McCauley MJ, Shokri L, Sefcikova J, Venclovas C, Beuning PJ, Williams MC - ACS Chem. Biol. (2008)

Binding isotherms for full-length protein and selected α constructs to DNA. a) Binding to dsDNA is determined by measuring the change in the melting force for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars represent the standard error from four experiments. Fits are to binding isotherms described within the text (eq 2 and eq 3), for n = 1 (solid lines) and n = 2 (dotted lines). For n = 1, Kds = 28 ± 7 × 106 M−1 for full-length α, 3.5 ± 0.9 × 106 M−1 for α1−917. and 0.4 ± 0.1 × 106 M−1 for α1−835. b) Binding to ssDNA is determined by measuring the fraction of ssDNA stabilized by protein binding, as found from eq 1, for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars are determined from errors in the fits. Fitting this data to the binding isotherms of the text (eq 3 and eq 4) determines Kss = 22 ± 6 × 106 M−1 for full-length α and 10 ± 3 × 106 M−1 for α917−1160. These results are summarized in Table 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2665888&req=5

fig8: Binding isotherms for full-length protein and selected α constructs to DNA. a) Binding to dsDNA is determined by measuring the change in the melting force for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars represent the standard error from four experiments. Fits are to binding isotherms described within the text (eq 2 and eq 3), for n = 1 (solid lines) and n = 2 (dotted lines). For n = 1, Kds = 28 ± 7 × 106 M−1 for full-length α, 3.5 ± 0.9 × 106 M−1 for α1−917. and 0.4 ± 0.1 × 106 M−1 for α1−835. b) Binding to ssDNA is determined by measuring the fraction of ssDNA stabilized by protein binding, as found from eq 1, for varying protein concentrations for full-length α (blue), the N-terminal constructs α1−917 (green) and α1−835 (yellow), and the C-terminal construct α917−1160 (red). Error bars are determined from errors in the fits. Fitting this data to the binding isotherms of the text (eq 3 and eq 4) determines Kss = 22 ± 6 × 106 M−1 for full-length α and 10 ± 3 × 106 M−1 for α917−1160. These results are summarized in Table 1.
Mentions: Stabilization of the double helix is shown in a series of extension (solid lines) and relaxation (dotted lines) cycles at varying salt concentrations. a, b) dsDNA is extended (black), and the helical form is disrupted at 62.6 ± 1.0 pN (32). As Na+ concentration is decreased, this transition occurs at lower forces and greater hysteresis is observed as DNA is relaxed (purple). As full-length protein is added (to the low-salt solution), the force required to disrupt the double helix increases again (blue). Furthermore, after a constant extension experiment in which the extension is fixed for 30 min, significant binding to the single strand appears (green), but only for the full-length protein (panel a) and not for the N-terminal α1−917 (panel b). c) The DNA melting force varies with Na+ concentration (purple), while melting force in the presence of 100 nM α and 500 nM α1−917 was observed to be independent of salt concentration. Uncertainties are determined from averages over three experiments. The average melting force for DNA, over the full range of Na+ concentrations studied, is 65.2 ± 0.4 pN in the presence of α protein and 65.1 ± 0.6 pN for α1−917. Higher concentrations of the α1−917 construct were necessary because of weaker binding (see Figure 5, panel b and Figure 8, panel a).

Bottom Line: In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force.From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA.The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Northeastern University, Boston, Massachusetts, 02115, USA.

ABSTRACT
The alpha subunit of the replicative DNA polymerase III of Escherichia coli is the active polymerase of the 10-subunit bacterial replicase. The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain. In a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded DNA, in distinct subdomains of the protein. The portion of the protein that binds to double-stranded DNA stabilizes the DNA helix, because protein binding must be at least partially disrupted with increasing force to melt DNA. Upon relaxation, the DNA fails to fully reanneal, because bound protein interferes with the reformation of the double helix. In addition, the single-stranded DNA binding component appears to be passive, as the protein does not facilitate melting but instead binds to single-stranded regions already separated by force. From DNA stretching measurements we determine equilibrium association constants for the binding of alpha and several fragments to dsDNA and ssDNA. The results demonstrate that ssDNA binding is localized to the C-terminal region that contains the OB-fold domain, while a tandem helix-hairpin-helix (HhH) 2 motif contributes significantly to dsDNA binding.

Show MeSH
Related in: MedlinePlus