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A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding.

Whelan F, Stead JA, Shkumatov AV, Svergun DI, Sanders CM, Antson AA - Nucleic Acids Res. (2011)

Bottom Line: Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis.We argue that these interactions impart processivity to DNA unwinding.Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

View Article: PubMed Central - PubMed

Affiliation: York Structural Biology Laboratory, The University of York, York YO10 5DD, UK.

ABSTRACT
The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

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Helicase domain enzymatic assays. (A) ATPase assays (4 µM HD protein) with or without T30 ssDNA oligonucleotide (1:1.5 T30:HD monomer) were sampled over time (2.5, 5, 10, 20 and 40 min) and phosphate release determined. Without T30 ssDNA, the ATPase activity of E1HDΔC26 was reduced ~50% relative to E1HD. With ssDNA, the activity of E1HDΔC26 was ~85% that of E1HD. Turnover numbers (per second) were determined from the slope of the graph using the values up to 20 min where ATP hydrolysis is in the linear range. (B) Helicase assays (0.1, 0.2, 0.3, 0.4 and 0.5 µM HD protein) were performed with substrates with a 55 base 3′ poly T tail and duplex portions of 25, 76 and 153 base (T55-ds25, T55-ds76 and T55-153; 0.5 nM each) combined in the same reaction. The short strand of each was end-labelled with 32P and reaction products were resolved by polyacrylamide gel electrophoresis. The line graph shows unwinding as a function of protein concentration. The bar graph shows the data for the intermediate concentration (0.3 µM). Compared to E1HD, unwinding of dsDNA by E1HDΔC26 was significantly and progressively impaired with increasing DNA length.
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gkr906-F2: Helicase domain enzymatic assays. (A) ATPase assays (4 µM HD protein) with or without T30 ssDNA oligonucleotide (1:1.5 T30:HD monomer) were sampled over time (2.5, 5, 10, 20 and 40 min) and phosphate release determined. Without T30 ssDNA, the ATPase activity of E1HDΔC26 was reduced ~50% relative to E1HD. With ssDNA, the activity of E1HDΔC26 was ~85% that of E1HD. Turnover numbers (per second) were determined from the slope of the graph using the values up to 20 min where ATP hydrolysis is in the linear range. (B) Helicase assays (0.1, 0.2, 0.3, 0.4 and 0.5 µM HD protein) were performed with substrates with a 55 base 3′ poly T tail and duplex portions of 25, 76 and 153 base (T55-ds25, T55-ds76 and T55-153; 0.5 nM each) combined in the same reaction. The short strand of each was end-labelled with 32P and reaction products were resolved by polyacrylamide gel electrophoresis. The line graph shows unwinding as a function of protein concentration. The bar graph shows the data for the intermediate concentration (0.3 µM). Compared to E1HD, unwinding of dsDNA by E1HDΔC26 was significantly and progressively impaired with increasing DNA length.

Mentions: The ATPase activity of E1HD and E1HDΔC26 was determined by measuring the release of 32Pi from [γ-32P]ATP, in the absence and presence of T30 ssDNA, over time (Figure 2A). Without the T30 oligonucleotide, ATP hydrolysis was reduced by ~50% for E1HDΔC26 compared to E1HD (the turnover numbers determined from initial rates are 0.29 and 0.14/s for E1HD and E1HDΔC26 respectively). In the presence of excess T30 ssDNA (1:1.5 T30:HD monomer), the rate of ATP hydrolysis increased by ~1.6-fold for E1HD. However, for E1HDΔC26 the increase was 2.9-fold (E1HD 0.47/s and E1HDΔC26 0.4/s determined from the initial rates) and therefore the difference in enzymatic activity between the two forms of the enzyme is small in the presence of ssDNA. These data reflect the data in Figure 1B and C above, where formation of the hexameric E1 assembly, the active form of the enzyme, is most efficient with ssDNA and ATP/Mg2+ combined. They also argue that the C-terminal 26 amino acids of E1HD have a significant indirect effect on ATP turnover by influencing the hexameric assembly state of the enzyme rather than the active site directly.Figure 2.


A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding.

Whelan F, Stead JA, Shkumatov AV, Svergun DI, Sanders CM, Antson AA - Nucleic Acids Res. (2011)

Helicase domain enzymatic assays. (A) ATPase assays (4 µM HD protein) with or without T30 ssDNA oligonucleotide (1:1.5 T30:HD monomer) were sampled over time (2.5, 5, 10, 20 and 40 min) and phosphate release determined. Without T30 ssDNA, the ATPase activity of E1HDΔC26 was reduced ~50% relative to E1HD. With ssDNA, the activity of E1HDΔC26 was ~85% that of E1HD. Turnover numbers (per second) were determined from the slope of the graph using the values up to 20 min where ATP hydrolysis is in the linear range. (B) Helicase assays (0.1, 0.2, 0.3, 0.4 and 0.5 µM HD protein) were performed with substrates with a 55 base 3′ poly T tail and duplex portions of 25, 76 and 153 base (T55-ds25, T55-ds76 and T55-153; 0.5 nM each) combined in the same reaction. The short strand of each was end-labelled with 32P and reaction products were resolved by polyacrylamide gel electrophoresis. The line graph shows unwinding as a function of protein concentration. The bar graph shows the data for the intermediate concentration (0.3 µM). Compared to E1HD, unwinding of dsDNA by E1HDΔC26 was significantly and progressively impaired with increasing DNA length.
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Related In: Results  -  Collection

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gkr906-F2: Helicase domain enzymatic assays. (A) ATPase assays (4 µM HD protein) with or without T30 ssDNA oligonucleotide (1:1.5 T30:HD monomer) were sampled over time (2.5, 5, 10, 20 and 40 min) and phosphate release determined. Without T30 ssDNA, the ATPase activity of E1HDΔC26 was reduced ~50% relative to E1HD. With ssDNA, the activity of E1HDΔC26 was ~85% that of E1HD. Turnover numbers (per second) were determined from the slope of the graph using the values up to 20 min where ATP hydrolysis is in the linear range. (B) Helicase assays (0.1, 0.2, 0.3, 0.4 and 0.5 µM HD protein) were performed with substrates with a 55 base 3′ poly T tail and duplex portions of 25, 76 and 153 base (T55-ds25, T55-ds76 and T55-153; 0.5 nM each) combined in the same reaction. The short strand of each was end-labelled with 32P and reaction products were resolved by polyacrylamide gel electrophoresis. The line graph shows unwinding as a function of protein concentration. The bar graph shows the data for the intermediate concentration (0.3 µM). Compared to E1HD, unwinding of dsDNA by E1HDΔC26 was significantly and progressively impaired with increasing DNA length.
Mentions: The ATPase activity of E1HD and E1HDΔC26 was determined by measuring the release of 32Pi from [γ-32P]ATP, in the absence and presence of T30 ssDNA, over time (Figure 2A). Without the T30 oligonucleotide, ATP hydrolysis was reduced by ~50% for E1HDΔC26 compared to E1HD (the turnover numbers determined from initial rates are 0.29 and 0.14/s for E1HD and E1HDΔC26 respectively). In the presence of excess T30 ssDNA (1:1.5 T30:HD monomer), the rate of ATP hydrolysis increased by ~1.6-fold for E1HD. However, for E1HDΔC26 the increase was 2.9-fold (E1HD 0.47/s and E1HDΔC26 0.4/s determined from the initial rates) and therefore the difference in enzymatic activity between the two forms of the enzyme is small in the presence of ssDNA. These data reflect the data in Figure 1B and C above, where formation of the hexameric E1 assembly, the active form of the enzyme, is most efficient with ssDNA and ATP/Mg2+ combined. They also argue that the C-terminal 26 amino acids of E1HD have a significant indirect effect on ATP turnover by influencing the hexameric assembly state of the enzyme rather than the active site directly.Figure 2.

Bottom Line: Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis.We argue that these interactions impart processivity to DNA unwinding.Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

View Article: PubMed Central - PubMed

Affiliation: York Structural Biology Laboratory, The University of York, York YO10 5DD, UK.

ABSTRACT
The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

Show MeSH