Limits...
Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly cy-dye labelled DNA.

Wynne SA, Pinheiro VB, Holliger P, Leslie AG - PLoS ONE (2013)

Bottom Line: The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu.In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand.Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.

ABSTRACT
Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.

Show MeSH
Polymerase activity at 60°C.A) The primer extension assays used a primer labelled with FAM (fluorescein) and unlabelled template. Sites of Cy5-dCTP incorporation (Gs in the template) are shown in red. B) Primer extension time course comparing wild-type Pfu(exo-) and engineered Pfu-E10 polymerases at 60°C. Extension times are shown in minutes. Extension products used to quantify extension beyond the seven consecutive Cy5-dCTP incorporations (C7 challenge) are highlighted in red – see Materials and Methods for details. C) Fraction of the primers extended beyond the C7 challenge for both tested polymerases – results are shown for two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3733885&req=5

pone-0070892-g001: Polymerase activity at 60°C.A) The primer extension assays used a primer labelled with FAM (fluorescein) and unlabelled template. Sites of Cy5-dCTP incorporation (Gs in the template) are shown in red. B) Primer extension time course comparing wild-type Pfu(exo-) and engineered Pfu-E10 polymerases at 60°C. Extension times are shown in minutes. Extension products used to quantify extension beyond the seven consecutive Cy5-dCTP incorporations (C7 challenge) are highlighted in red – see Materials and Methods for details. C) Fraction of the primers extended beyond the C7 challenge for both tested polymerases – results are shown for two independent experiments.

Mentions: Although many natural polymerases are capable of efficient incorporation of and extension from a single Cy3 or Cy5-modified nucleotide, the synthetic challenge rapidly escalates at higher incorporation densities due to the steric bulk (and potentially other effects) of the large, hydrophobic cyanine dye heterocycles clustering in the major groove. This has hindered synthesis of DNA with high-density arrays of Cy-dyes and the applications this might enable and has spurred the development of a dedicated polymerase Pfu-E10 [19], [20]. Indeed, primer extension assays from challenging templates (that require multiple consecutive incorporations of the modified nucleotide) demonstrate the extent to which the evolved polymerase (Pfu-E10) outperforms the wild-type (Pfu(exo-)) polymerase (Figure 1, Figure S1). Here the primer is labelled with fluorescein amidite (FAM) so that it can be visualised regardless of cyanine dye incorporation. In both conditions, the engineered polymerase Pfu-E10 readily synthesises full-length product traversing a dG7 template stretch requiring the consecutive incorporation of seven Cy5-dCTPs within the experimental time frame. In contrast, the wild-type Pfu exo- polymerase cannot traverse that stretch and stalls after incorporation of maximally five consecutive Cy5-dCTPs (with a major pause at n+3).


Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly cy-dye labelled DNA.

Wynne SA, Pinheiro VB, Holliger P, Leslie AG - PLoS ONE (2013)

Polymerase activity at 60°C.A) The primer extension assays used a primer labelled with FAM (fluorescein) and unlabelled template. Sites of Cy5-dCTP incorporation (Gs in the template) are shown in red. B) Primer extension time course comparing wild-type Pfu(exo-) and engineered Pfu-E10 polymerases at 60°C. Extension times are shown in minutes. Extension products used to quantify extension beyond the seven consecutive Cy5-dCTP incorporations (C7 challenge) are highlighted in red – see Materials and Methods for details. C) Fraction of the primers extended beyond the C7 challenge for both tested polymerases – results are shown for two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733885&req=5

pone-0070892-g001: Polymerase activity at 60°C.A) The primer extension assays used a primer labelled with FAM (fluorescein) and unlabelled template. Sites of Cy5-dCTP incorporation (Gs in the template) are shown in red. B) Primer extension time course comparing wild-type Pfu(exo-) and engineered Pfu-E10 polymerases at 60°C. Extension times are shown in minutes. Extension products used to quantify extension beyond the seven consecutive Cy5-dCTP incorporations (C7 challenge) are highlighted in red – see Materials and Methods for details. C) Fraction of the primers extended beyond the C7 challenge for both tested polymerases – results are shown for two independent experiments.
Mentions: Although many natural polymerases are capable of efficient incorporation of and extension from a single Cy3 or Cy5-modified nucleotide, the synthetic challenge rapidly escalates at higher incorporation densities due to the steric bulk (and potentially other effects) of the large, hydrophobic cyanine dye heterocycles clustering in the major groove. This has hindered synthesis of DNA with high-density arrays of Cy-dyes and the applications this might enable and has spurred the development of a dedicated polymerase Pfu-E10 [19], [20]. Indeed, primer extension assays from challenging templates (that require multiple consecutive incorporations of the modified nucleotide) demonstrate the extent to which the evolved polymerase (Pfu-E10) outperforms the wild-type (Pfu(exo-)) polymerase (Figure 1, Figure S1). Here the primer is labelled with fluorescein amidite (FAM) so that it can be visualised regardless of cyanine dye incorporation. In both conditions, the engineered polymerase Pfu-E10 readily synthesises full-length product traversing a dG7 template stretch requiring the consecutive incorporation of seven Cy5-dCTPs within the experimental time frame. In contrast, the wild-type Pfu exo- polymerase cannot traverse that stretch and stalls after incorporation of maximally five consecutive Cy5-dCTPs (with a major pause at n+3).

Bottom Line: The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu.In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand.Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.

ABSTRACT
Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.

Show MeSH