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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

Binding of the N-terminal constructs α1−917 (panel a) and α1−835 (panel b) shows no stabilization of ssDNA through the sequence of extension/relaxation cycles (in the sequence order blue, green, red), as indicated by the complete reannealing of DNA at high forces upon relaxation in both cases. A slight change in the melting force, indicating some dsDNA binding, is evident for α1−917 but not for α1−835. The strong overlap of the extension curves (solid lines) emphasizes the reproducibility of the data, whereas some variability is evident upon relaxation (dotted lines), though this is typical of these experiments with DNA even in the absence of protein (black).
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fig7: Binding of the N-terminal constructs α1−917 (panel a) and α1−835 (panel b) shows no stabilization of ssDNA through the sequence of extension/relaxation cycles (in the sequence order blue, green, red), as indicated by the complete reannealing of DNA at high forces upon relaxation in both cases. A slight change in the melting force, indicating some dsDNA binding, is evident for α1−917 but not for α1−835. The strong overlap of the extension curves (solid lines) emphasizes the reproducibility of the data, whereas some variability is evident upon relaxation (dotted lines), though this is typical of these experiments with DNA even in the absence of protein (black).

Mentions: N-terminal constructs consisting of either residues 1−917 (including the polymerase core and the HhH motifs) or 1−835 (lacking the tandem HhH motifs) showed no evidence of binding to ssDNA (Figure 7), as complete DNA reannealing occurs at high forces (∼40 pN) upon relaxation. Thus our observations allow us to rule out ssDNA binding by the (HhH)2 motifs or other regions of the N-terminus of α. Furthermore, the α1−917 construct shows substantial binding to dsDNA. However, the α1−835 construct does not show strong dsDNA binding, and therefore the tandem HhH motifs contribute significantly to dsDNA binding.


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 of the N-terminal constructs α1−917 (panel a) and α1−835 (panel b) shows no stabilization of ssDNA through the sequence of extension/relaxation cycles (in the sequence order blue, green, red), as indicated by the complete reannealing of DNA at high forces upon relaxation in both cases. A slight change in the melting force, indicating some dsDNA binding, is evident for α1−917 but not for α1−835. The strong overlap of the extension curves (solid lines) emphasizes the reproducibility of the data, whereas some variability is evident upon relaxation (dotted lines), though this is typical of these experiments with DNA even in the absence of protein (black).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Binding of the N-terminal constructs α1−917 (panel a) and α1−835 (panel b) shows no stabilization of ssDNA through the sequence of extension/relaxation cycles (in the sequence order blue, green, red), as indicated by the complete reannealing of DNA at high forces upon relaxation in both cases. A slight change in the melting force, indicating some dsDNA binding, is evident for α1−917 but not for α1−835. The strong overlap of the extension curves (solid lines) emphasizes the reproducibility of the data, whereas some variability is evident upon relaxation (dotted lines), though this is typical of these experiments with DNA even in the absence of protein (black).
Mentions: N-terminal constructs consisting of either residues 1−917 (including the polymerase core and the HhH motifs) or 1−835 (lacking the tandem HhH motifs) showed no evidence of binding to ssDNA (Figure 7), as complete DNA reannealing occurs at high forces (∼40 pN) upon relaxation. Thus our observations allow us to rule out ssDNA binding by the (HhH)2 motifs or other regions of the N-terminus of α. Furthermore, the α1−917 construct shows substantial binding to dsDNA. However, the α1−835 construct does not show strong dsDNA binding, and therefore the tandem HhH motifs contribute significantly to dsDNA binding.

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