Fig 1.
Comparison of the B-family DNA pols KOD and δ.
The domains are color coded as followed: the N-terminal (blue), exonuclease (magenta), finger (yellow), palm (cyan) and thumb (green) domain. The p/t complex is shown in grey. The polymerase active site and the exonuclease active site are indicated by arrows. The bound dNTP as well as the metal ions are shown as sticks and spheres, respectively.
Fig 2.
Overview of the closed ternary complex of KOD pol.
(A) The ternary complex is color coded as seen in Fig 1, the finger domain of the ternary complex (yellow) is closed by approximately 24° compared to the finger domain (orange) of the superimposed binary KOD complex (grey). (B) The electron density of the primer/template is shown at 1 σ as a blue mesh, the omit map at 3 σ of the dATP is shown as a pink mesh, the electron density of the Mg2+ and the two Mn2+ ions is shown as a blue mesh at 1 σ with the anomalous signal at 3 σ as a green mesh for the two Mn2+ ions. (C) The electron density for the finger domain at 1 σ is shown as a blue mesh.
Fig 3.
KOD DNA pol’s active site with bound dATP.
(A) The metal ions are coordinated by residues of the palm domain (cyan). Metal ion A (Mg2+, green) is coordinated by two water molecules, D542, the α-phosphate of dATP (pink) and D404. Metal ion B (Mn2+, purple) is coordinated by the α-, β- and γ- phosphate, D404, F405 and D542. Metal ion C (Mn2+, purple) is coordinated by the γ-phosphate, E580, F405, D404 and three water molecules, whereof one molecule is coordinated by E578, one by E580 and one by K464 (yellow) of the finger domain. The dATP makes further direct contacts with conserved residues of the finger domain (yellow), N491, K487 and R460 as well as water mediated contacts to Q483 and K464. Additionally, a water mediated interaction with the not conserved Q461 (yellow) can be formed. (B, C) DNA pols’ protein surface is shown in grey, the template in blue, the primer in bright blue, the bound adenosine triphosphate in pink with the N7 atom indicated as a blue sphere; (B) KOD DNA pol shows the N7 atom pointing towards a wide open crevice between the finger (yellow), palm and exonuclease domain; (C) KlenTaq DNA pol shows the N7 atom pointing towards the O-helix of the finger domain (yellow) and the thumb domain, the crevice overall being narrower compared to KOD DNA pol.
Fig 4.
Interaction pattern of KOD DNA pol, according to the strength of the interaction.
The interactions between the enzyme and the respective dNTP as well as between the enzyme and the template/ primer strand were assigned according to their strengths (see legend) (A) The interaction between the protein and the template and primer strand are shown. (B) The interactions with the dATP are shown, the stacking between Y409 and the sugar moiety is indicated by a dashed line.
Fig 5.
Electropotential map of KOD and KTQ DNA pols.
The electropotential is shown from +6 (red) to -6 (blue) kBT/e (T = 310 K). The primer is shown in pink, the template in violet and the dNTP is shown as yellow sticks. KOD DNA pol exhibits a long crevice between the thumb and palm domain reaching up along the β-hairpin to the N-terminal domain, in which the single stranded template may bind. This crevice is missing in KlenTaq DNA pol, where the single stranded template leaves the polymerase between the thumb and finger domain.
Fig 6.
The channel volumes for KOD and KlenTaq DNA pols.
The channel volumes were calculated with 3V algorithm for KOD (A and B) and KlenTaq (C and D) DNA pols, respectively. The protein is shown as grey surface, the primer in cyan and the template in blue. The bound dNTP is shown in magenta. A and C show the location within the enzyme, B and D the channels with respect to the DNA and triphosphate.