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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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Catalytic α subunit of E. coli replicative DNA pol III. a) Domain architecture of α subunit and five additional constructs studied in this work. Polymerase core indicates palm, thumb, and fingers domains; HhH denotes helix-hairpin-helix DNA-binding motif; β-binding motif denotes internal canonical and C-terminal β-binding motif; and OB-domain denotes predicted OB-fold domain. Residue numbers at the boundaries of major domains and at the ends of the fragments are noted according to ref (8). b) Homology model of the OB-fold domain, encompassing residues 978−1078 and derived as described in the text. ssDNA is predicted to interact with the highly conserved residue F1031 shown in the figure. The coordinates for the model are available at http://www.ibt.lt/bioinformatics/models/polIII_OB. (c) dsDNA binds nonspecifically to the tandem HhH motif in this model of residues 833−889.
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fig1: Catalytic α subunit of E. coli replicative DNA pol III. a) Domain architecture of α subunit and five additional constructs studied in this work. Polymerase core indicates palm, thumb, and fingers domains; HhH denotes helix-hairpin-helix DNA-binding motif; β-binding motif denotes internal canonical and C-terminal β-binding motif; and OB-domain denotes predicted OB-fold domain. Residue numbers at the boundaries of major domains and at the ends of the fragments are noted according to ref (8). b) Homology model of the OB-fold domain, encompassing residues 978−1078 and derived as described in the text. ssDNA is predicted to interact with the highly conserved residue F1031 shown in the figure. The coordinates for the model are available at http://www.ibt.lt/bioinformatics/models/polIII_OB. (c) dsDNA binds nonspecifically to the tandem HhH motif in this model of residues 833−889.

Mentions: The 10-subunit DNA polymerase III holoenzyme has been shown to bind approximately 30 nucleotides of double-stranded DNA in a primer:template complex (12). Pol III α subunit has at least two distinct domains that may be important for binding DNA (Figure 1). A “helix-hairpin-helix” (HhH) motif, initially predicted in the α subunit by sequence analysis (13), is a widespread motif involved in non-sequence-specific binding of either ds- or ss-DNA. Crystal structures of the Pol III α subunits of E. coli(8) and T. aquaticus(9) confirm the presence of the HhH motif slightly N-terminal to the internal β-processivity clamp binding motif. The HhH motif in both structures may be considered as part of a distinct (HhH)2 domain formed by two consecutively duplicated HhH motifs (14). (HhH)2 domains are present in a majority of HhH-containing proteins and provide a symmetric way of binding to dsDNA, as in DNA polymerase β (14). HhH domains are also known to mediate protein−protein interactions (15). The second putative DNA binding domain in the α subunit is an oligonucleotide/oligosaccharide binding (OB-fold) domain (16,17). The OB-fold domain is located near the C-terminus (7–9,18), which is not present in the E. coli structure. OB-domains are functionally diverse, as they are involved in binding oligonucleotides, oligosaccharides, or metal ions and also in mediating protein−protein interactions (18,19). Thus, pol III possesses two domains outside of the polymerase core that are often associated with DNA binding. Yet the DNA binding activity of either domain has not been directly demonstrated.


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)

Catalytic α subunit of E. coli replicative DNA pol III. a) Domain architecture of α subunit and five additional constructs studied in this work. Polymerase core indicates palm, thumb, and fingers domains; HhH denotes helix-hairpin-helix DNA-binding motif; β-binding motif denotes internal canonical and C-terminal β-binding motif; and OB-domain denotes predicted OB-fold domain. Residue numbers at the boundaries of major domains and at the ends of the fragments are noted according to ref (8). b) Homology model of the OB-fold domain, encompassing residues 978−1078 and derived as described in the text. ssDNA is predicted to interact with the highly conserved residue F1031 shown in the figure. The coordinates for the model are available at http://www.ibt.lt/bioinformatics/models/polIII_OB. (c) dsDNA binds nonspecifically to the tandem HhH motif in this model of residues 833−889.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Catalytic α subunit of E. coli replicative DNA pol III. a) Domain architecture of α subunit and five additional constructs studied in this work. Polymerase core indicates palm, thumb, and fingers domains; HhH denotes helix-hairpin-helix DNA-binding motif; β-binding motif denotes internal canonical and C-terminal β-binding motif; and OB-domain denotes predicted OB-fold domain. Residue numbers at the boundaries of major domains and at the ends of the fragments are noted according to ref (8). b) Homology model of the OB-fold domain, encompassing residues 978−1078 and derived as described in the text. ssDNA is predicted to interact with the highly conserved residue F1031 shown in the figure. The coordinates for the model are available at http://www.ibt.lt/bioinformatics/models/polIII_OB. (c) dsDNA binds nonspecifically to the tandem HhH motif in this model of residues 833−889.
Mentions: The 10-subunit DNA polymerase III holoenzyme has been shown to bind approximately 30 nucleotides of double-stranded DNA in a primer:template complex (12). Pol III α subunit has at least two distinct domains that may be important for binding DNA (Figure 1). A “helix-hairpin-helix” (HhH) motif, initially predicted in the α subunit by sequence analysis (13), is a widespread motif involved in non-sequence-specific binding of either ds- or ss-DNA. Crystal structures of the Pol III α subunits of E. coli(8) and T. aquaticus(9) confirm the presence of the HhH motif slightly N-terminal to the internal β-processivity clamp binding motif. The HhH motif in both structures may be considered as part of a distinct (HhH)2 domain formed by two consecutively duplicated HhH motifs (14). (HhH)2 domains are present in a majority of HhH-containing proteins and provide a symmetric way of binding to dsDNA, as in DNA polymerase β (14). HhH domains are also known to mediate protein−protein interactions (15). The second putative DNA binding domain in the α subunit is an oligonucleotide/oligosaccharide binding (OB-fold) domain (16,17). The OB-fold domain is located near the C-terminus (7–9,18), which is not present in the E. coli structure. OB-domains are functionally diverse, as they are involved in binding oligonucleotides, oligosaccharides, or metal ions and also in mediating protein−protein interactions (18,19). Thus, pol III possesses two domains outside of the polymerase core that are often associated with DNA binding. Yet the DNA binding activity of either domain has not been directly demonstrated.

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