Snapshot of a DNA Polymerase while Incorporating Two Consecutive C5-Modified Nucleotides

Functional nucleotides are important in many cutting-edge biomolecular techniques. Often several modified nucleotides have to be incorporated consecutively. This structural study of KlenTaq DNA polymerase, a truncated form of <i>Thermus aquaticus</i> DNA polymerase, gives first insights how multiple modifications are processed by a DNA polymerase and, therefore, contribute to the understanding of these enzymes in their interplay with artificial substrates

Crystal structures of ternary complexes of archaeal B-family DNA polymerases

<div><p>Archaeal B-family polymerases drive biotechnology by accepting a wide substrate range of chemically modified nucleotides. By now no structural data for archaeal B-family DNA polymerases in a closed, ternary complex are available, which would be the basis for developing next generation nucleotides. We present the ternary crystal structures of KOD and 9°N DNA polymerases complexed with DNA and the incoming dATP. The structures reveal a third metal ion in the active site, which was so far only observed for the eukaryotic B-family DNA polymerase δ and no other B-family DNA polymerase. The structures reveal a wide inner channel and numerous interactions with the template strand that provide space for modifications within the enzyme and may account for the high processivity, respectively. The crystal structures provide insights into the superiority over other DNA polymerases concerning the acceptance of modified nucleotides.</p></div

Electropotential map of KOD and KTQ DNA pols.

<p>The electropotential is shown from +6 (red) to -6 (blue) k_BT/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.</p

KOD DNA pol’s active site with bound dATP.

<p>(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 <i>N</i>7 atom indicated as a blue sphere; (B) KOD DNA pol shows the <i>N</i>7 atom pointing towards a wide open crevice between the finger (yellow), palm and exonuclease domain; (C) KlenTaq DNA pol shows the <i>N</i>7 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.</p

Overview of the closed ternary complex of KOD pol.

<p>(A) The ternary complex is color coded as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188005#pone.0188005.g001" target="_blank">Fig 1</a>, 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.</p

The channel volumes for KOD and KlenTaq DNA pols.

<p>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.</p

Interaction pattern of KOD DNA pol, according to the strength of the interaction.

<p>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.</p

Chorismatase Mechanisms Reveal Fundamentally Different Types of Reaction in a Single Conserved Protein Fold

Chorismatases are a class of chorismate-converting enzymes involved in the biosynthetic pathways of different natural products, many of them with interesting pharmaceutical characteristics. So far, three subfamilies of chorismatases are described that convert chorismate into different (dihydro-)benzoate derivatives (CH-FkbO, CH-Hyg5, and CH-XanB2). Until now, the detailed enzyme mechanism and the molecular basis for the different reaction products were unknown. Here we show that the CH-FkbO and CH-Hyg5 subfamilies share the same protein fold, but employ fundamentally different reaction mechanisms. While the FkbO reaction is a typical hydrolysis, the Hyg5 reaction proceeds intramolecularly, most likely via an arene oxide intermediate. Two nonconserved active site residues were identified that are responsible for the different reaction mechanisms in CH-FkbO and CH-Hyg5. Further, we propose an additional amino acid residue to be responsible for the discrimination of the CH-XanB2 subfamily, which catalyzes the formation of two different hydroxybenzoate regioisomers, likely in a single active site. A multiple sequence alignment shows that these three crucial amino acid positions are located in conserved motifs and can therefore be used to assign unknown chorismatases to the corresponding subfamily

Structures of <i>KlenTaq</i> DNA Polymerase Caught While Incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates

The capability of DNA polymerases to accept chemically modified nucleotides is of paramount importance for many biotechnological applications. Although these analogues are widely used, the structural basis for the acceptance of the unnatural nucleotide surrogates has been only sparsely explored. Here we present in total six crystal structures of modified 2′-deoxynucleoside-5′-<i>O</i>-triphosphates (dNTPs) carrying modifications at the C5 positions of pyrimidines or C7 positions of 7-deazapurines in complex with a DNA polymerase and a primer/template complex. The modified dNTPs are in positions poised for catalysis leading to incorporation. These structural data provide insight into the mechanism of incorporation and acceptance of modified dNTPs. Our results open the door for rational design of modified nucleotides, which should offer great opportunities for future applications

Supplementary Materials from PK1 from <i>Drosophila</i> obscurin is an inactive pseudokinase with scaffolding properties

Figures and Text Supplementary to the main tex