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

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

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

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

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

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

Structural Insights into DNA Replication without Hydrogen Bonds

The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d<b>5SICS</b>-d<b>NaM</b> is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d<b>5SICS</b>TP opposite d<b>NaM</b> proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson–Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of d<b>NaM</b>TP opposite d<b>5SICS</b>, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5<b>SICS</b>TP opposite d<b>NaM</b>, addition of d<b>NaM</b>TP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation