Figure 1.
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.
Figure 2.
Summary of primer extension assays.
A) Key primer extension reactions visualised via the FAM channel. Only engineered E10 polymerase is capable of extending the FAM-labelled primer to full-length when incorporating Cy5-dCTP. No template-independent extension is observed in the absence of dCTP (dDTP only ie dGTP, dTTP and dATP) and both enzymes can synthesise the template with the natural dCTP substrate (dNTP). The discrepancy in migration between primers extended with natural triphosphates and with Cy5-dCTP, is due to the significant increase in monomer mass introduced by the Cy5 moiety. B) The same reactions visualised by the Cy5 channel to see only Cy5-labelled primer. It is clear that Cy5 signals are only observed after the incorporation of a Cy5-dCTP and co-localise to the signals observed in the FAM channel. C) Overlay of A and B in colour.
Figure 3.
Structure of the Pfu-E10 polymerase.
Cartoon representation of the apo polymerase structure with domains coloured as follows: N-terminal domain: red, exonuclease domain: gold, linker region: yellow, palm domain: pale blue, fingers domain: green and thumb domain: dark blue. The side chains of the mutated residues (V93Q, D141A, E143A, V337I, E399D, N400D, R407I and Y546H) are shown with atoms represented as red spheres.
Table 1.
Data collection and refinement statistics.
Table 2.
Structural similarity of Apo Pfu-E10 with other polymerases.
Figure 4.
Structure of the Pfu-E10:DNA binary complex.
Cartoon representation of the binary complex. Polymerase domains are coloured as in Figure 3. The DNA primer strand is shown in grey, the template strand in magenta.
Figure 5.
Density for the DNA and active site in the Pfu-E10:DNA binary complex.
An omit map was calculated omitting the co-ordinates of the DNA. This map, contoured at 1.25 sigma is shown in pale blue, with the DNA primer and terminal dideoxy cytidine (DOC) in grey, the DNA template in magenta, and Pfu:E10 residues in dark blue.
Figure 6.
Protein-DNA interactions in the Pfu-E10 binary complex.
Schematic representation of the protein DNA-contacts. Brackets indicate that the interaction is via the main chain peptide rather than the side chain, with residues coloured by domain with palm domain: pale blue, fingers domain: green and thumb domain: dark blue. Most interactions are with the sugar-phosphate backbone, however residues in the centre of the figure indicate a minor groove interaction. Full details are given in the text. The three residues at the 5′ end of the template strand could not be modelled.
Figure 7.
Comparison of the apo and binary structures of the Pfu-E10 polymerase.
The apo structure (gold) is shown following superposition of residues in the palm domain on the binary complex (blue). The large movement of the thumb domain is apparent. Residues are labelled in gold for the apo polymerase and in blue for the binary structure.
Figure 8.
Comparison of the Pfu-E10:DNA binary structure with RB69:DNA:dTTP ternary complex.
Superposition of the Pfu-E10:DNA binary structure (blue) onto palm domain residues of the RB69:DNA:dTTP ternary complex (green) (Pfu-E10 residues 384–414, RB69 390–420). DNA from Pfu:E10 complex is shown with grey primer and magenta template. The DNA strands of both structures and many of their domains aligned. The fingers domains show the greatest difference, the RB69 fingers have closed around a dNTP, whereas the Pfu:E10 fingers have not.
Table 3.
Structural similarity of Binary E10:DNA complex with RB69 ternary complex.
Figure 9.
Model of a possible ternary complex of Pfu:E10: DNA:Cy5-dCTP.
A). Each domain from the binary PFu:E10:DNA structure was superimposed separately onto the corresponding domain from the RB69:DNA:dTTP ternary complex, and coloured as for Figure 3. A molecule of Cy5-dCTP was then modelled into this with green carbon, red oxygen, blue nitrogen, yellow sulphur and purple phosphorous atoms, to try to understand how such a large molecule could fit into the complex. There appears to be a channel between the Fingers, Thumb and Exonuclease domains into which the large Cy5 molecule could extend. B). Representation of Cy5-dCTP.