Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA

Samantha A Wynne,Andrew G W Leslie,Vitor B Pinheiro,Philipp Holliger,Giovanni Maga

doi:10.1371/journal.pone.0070892

Samantha A Wynne, Andrew G W Leslie + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0070892

Copy DOI

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.

Highlights

Sequence analysis has identified six different families of DNAdependent DNA polymerases (A, B, C, D, X and Y) [1], [2]
CyDNA Synthesis by the E10 Polymerase 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 of the large, hydrophobic cyanine dye heterocycles clustering in the major groove
The engineered polymerase Pyrococcus furiosus (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

Summary

Introduction

Sequence analysis has identified six different families of DNAdependent DNA polymerases (A, B, C, D, X and Y) [1], [2]. High resolution structures of apo, binary and ternary complexes have been obtained for KlenTaq (polA) [4], [5], RB69 polymerase (polB) [6], [7], E. coli polIII (polC) [8], [9], Dpo (polY) [10], and polbeta (polX) [11]. While there are several high-resolution structures of the apo-forms of biotechnologically important polB family polymerases from hyperthermophilic archea (Thermococcus gorgonarius (Tgo) [12], Thermococcus sp. The replicative DNA polymerase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) is a member of that polB family and is used extensively in biotechnology applications including high-fidelity PCR. The resulting variant E10, derived from an exonuclease-deficient version of Pfu polymerase (with mutations V93Q, D141A and E143A) and with additional mutations in the conserved A- (V337I, E399D, N400D, R407I) and C- active site sequence motifs (Y546H) was able to efficiently PCR DNA while completely replacing all dCTP on both strands with cyanine labelled dCTP (Cy3-dCTP or Cy5-dCTP) giving rise to highlyfluorescent products, termed Cy-DNA [19] with applications in advanced microscopy applications [20]

Methods

Results

Conclusion