S.A.Wynne
et al.
(2013).
Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly cy-dye labelled DNA.
Plos One,
8,
e70892.
PubMed id: 23940661
DOI: 10.1371/journal.pone.0070892
Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly cy-dye labelled DNA.
S.A.Wynne,
V.B.Pinheiro,
P.Holliger,
A.G.Leslie.
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.
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