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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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Related in: MedlinePlus

Dual beam optical tweezers experiment schematic with accompanying microscope images. a) Streptavidin-coated beads (5.5 μm diameter) are fixed upon a micropipette tip and held within a dual beam confocal trap (∼1 μm diameter, which appears as the bright spot in the accompanying photograph). Biotinylated phage λ-DNA (16.5 μm contour length) is extended between these beads, and the subsequent increase in tension is determined by the deflection of the trapping lasers upon a pair of photodiode detectors. b) Solutions of varying concentrations of buffer, salt, and DNA binding protein (shown in green and yellow) are introduced. c) Measuring the force required for DNA extension in the presence of protein reveals information about binding to the double strand. d) Force-induced DNA melting (and subsequent relaxation) allows protein binding to ssDNA, which is observed as a change in the measured length.
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fig2: Dual beam optical tweezers experiment schematic with accompanying microscope images. a) Streptavidin-coated beads (5.5 μm diameter) are fixed upon a micropipette tip and held within a dual beam confocal trap (∼1 μm diameter, which appears as the bright spot in the accompanying photograph). Biotinylated phage λ-DNA (16.5 μm contour length) is extended between these beads, and the subsequent increase in tension is determined by the deflection of the trapping lasers upon a pair of photodiode detectors. b) Solutions of varying concentrations of buffer, salt, and DNA binding protein (shown in green and yellow) are introduced. c) Measuring the force required for DNA extension in the presence of protein reveals information about binding to the double strand. d) Force-induced DNA melting (and subsequent relaxation) allows protein binding to ssDNA, which is observed as a change in the measured length.

Mentions: Force spectroscopy experiments (Figure 2) probe the elasticity of dsDNA and convert it into ssDNA at a critical melting force (Figure 3). These experiments may be repeated in the presence of DNA binding ligands. Whereas binding to dsDNA is observed to stabilize the helical form, binding to ssDNA destabilizes the double helix and is manifested in the data as a decrease in either the melting force or the length of the overstretching plateau (24–29). Thus monitoring the force versus length of a cycle of DNA extension and relaxation serves to assay double- and single-stranded DNA binding. Additional experimental details are included in Supporting Information.


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)

Dual beam optical tweezers experiment schematic with accompanying microscope images. a) Streptavidin-coated beads (5.5 μm diameter) are fixed upon a micropipette tip and held within a dual beam confocal trap (∼1 μm diameter, which appears as the bright spot in the accompanying photograph). Biotinylated phage λ-DNA (16.5 μm contour length) is extended between these beads, and the subsequent increase in tension is determined by the deflection of the trapping lasers upon a pair of photodiode detectors. b) Solutions of varying concentrations of buffer, salt, and DNA binding protein (shown in green and yellow) are introduced. c) Measuring the force required for DNA extension in the presence of protein reveals information about binding to the double strand. d) Force-induced DNA melting (and subsequent relaxation) allows protein binding to ssDNA, which is observed as a change in the measured length.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665888&req=5

fig2: Dual beam optical tweezers experiment schematic with accompanying microscope images. a) Streptavidin-coated beads (5.5 μm diameter) are fixed upon a micropipette tip and held within a dual beam confocal trap (∼1 μm diameter, which appears as the bright spot in the accompanying photograph). Biotinylated phage λ-DNA (16.5 μm contour length) is extended between these beads, and the subsequent increase in tension is determined by the deflection of the trapping lasers upon a pair of photodiode detectors. b) Solutions of varying concentrations of buffer, salt, and DNA binding protein (shown in green and yellow) are introduced. c) Measuring the force required for DNA extension in the presence of protein reveals information about binding to the double strand. d) Force-induced DNA melting (and subsequent relaxation) allows protein binding to ssDNA, which is observed as a change in the measured length.
Mentions: Force spectroscopy experiments (Figure 2) probe the elasticity of dsDNA and convert it into ssDNA at a critical melting force (Figure 3). These experiments may be repeated in the presence of DNA binding ligands. Whereas binding to dsDNA is observed to stabilize the helical form, binding to ssDNA destabilizes the double helix and is manifested in the data as a decrease in either the melting force or the length of the overstretching plateau (24–29). Thus monitoring the force versus length of a cycle of DNA extension and relaxation serves to assay double- and single-stranded DNA binding. Additional experimental details are included in Supporting Information.

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